| Background and objectiveIn the last thirty years, human assisted reproductive technology(ART) have been developed rapidly.But to date, the implantation rate per cycle has still been close to 20%. In addition,only approximately 30%-40% of the clinical pregnancy rate per (IVF) cycle and approximately 25% of living birth rate per IVF cycle have been observed in women who received assisted reproductive techniques. One of the important reasons is known to be the immture and abnormal oocytes collected in the superovulatary environment during the controlled ovarian hyperstimulation(COH) procedure, resulting in low fertilization rate and poor quality embryo. How to get mature oocyte with high quality and development potential remains one of the major goals in the field of assisted reproduction. On the other hand, to date we evaluate and select the quality and competence of oocyte and early embryo by morphological criteria in ART laboratory. The morphological appearance of the oocyte and embryo, however, does not accurately predict its competence.The morphological criteria is not unified, and it depend on staff skills and experiences. We lack of a more effective role in the detection methods to assess the development potential the oocyte. We can get a quite number of cumulus cells when oocyte was retrived, even more they are abandoned before ICSI. Cumulus cell is a suitable candidate in which we can look for some biochemical markers for oocyte maturation and competence. Further research on these biochemical markers may helps to reveal the mechanism of follicular development and oocyte maturation, and may provide some new ideas for improving ovarian stimulation protocol and in vitro culture system in ART laboratory.Cumulus cells is defined as a group of closely associated granulose cells, which surrounds the oocyte in antral ovarian follicle. Cumulus cells communicate with each other and with the oocyte by means of gap junctions through which cumulus cells play an important role in oocyte maturation. The fully developed cumulus cells exert at least three important biological functions:the first is to keep the oocyte under meiotic arrest, the second is to participate in the induction of oocyte meiosis resume at the time of ovulation, and the third is to support cytoplasmic maturation of the oocyte. In spite of numerous data on the interaction and dialogue between cumulus cell and oocyte, the molecular mechanism involved in this process needs to be clarified.Protein is the performer of biological processes. With the development of proteomics technology, it is feasible to study whole changes of proteome in the physiological and pathological process now. At present there are many novel differential proteomics analysis methods, among which the two-dimensional difference gel electrophoresis (2D-DIGE) technology becomes one of the most popular one. Because of the application of the internal standard,2D-DIGE not only have the high-resolution feature, inherited from two-dimensional gel electrophoresis (2-DE), but also have high reproducibility, high sensitivity, high throughput and high dynamic range.So far there have been only a limit number of proteomic research about ovarian reproductive cell and somatic cells, and lack of proteomic research focused on the relation of cumulus cells and oocyte maturation. In this paper, we collected cumulus cells from the women who undergoing ICSI treatment and subjected to controlled ovarian hyperstimulation, used 2D-DIGE techlonogy, MELDI-TOF-MS and bioinformatics, studied on the differential expressed profiles of human cumulus cells during oocyte maturation. Our aim was therefore to look for oocyte maturation-related proteins in human cumulus cells, to provide the molecular basis for our understanding of the cumulus cells roles in oocyte maturation, and to provide some potential biochemical markers for oocyte quality assessment and selection in ART laboratory.Chapter 1:Differential Proteomic Study on cumulus cells around human mature and immature oocytesObjectiveIn order to obtain the oocyte maturation-related proteins in human cumulus cells, the protein expressed profiles of human cumulus cells surrounding muture and immature oocytes were compared using 2D-DIGE quantitative proteomics, and then differential protein spots with statistical significant were identified by MALDI-TOF-MS and analyzed by bioinformatics.Methods1. SamplesThe 89 subjects recruited in this study were undergoing ICSI for male factor reason at Nanfang Hospital Reproductive Center from June to December in 2009.The patients were subjected to controlled ovarian hyperstimulation(COH) accomplished with a modified long luteal suppression protocol.Cumulus cells were mechanically removed from oocytes on the day of egg retrieval and grouped by the oocyte maturation:Mature group:Cumulus cells surrounding MⅡoocyte;Immature group:Cumulus cells surrounding M I and GV oocyte.2. Two dimensional-difference in gel electrophoresis(2D-DIGE)Total protein was extracted, purified and quantified, and then separated by 2D-DIGE, Expression maps of different proteins were taken from protein spots signed by Cydye in advance in the 2D-DIGE gel after laser passing by distinct wavelength using multifunctional scanner, and then analyzed by DeCyder. Preparative gels were stained with Coomassie brilliant blue and scanned.3. Identification of differential protein by MELDI-TOF-MS.Preparative gels corresponding with 2D-DIGE were inserted and fixed in Ettan Spot Handling Workstation. Spots with differential rate of more than 1.5 were distected, enzymatic hydrolysis and spotted in mass spectrometry. Peptide mass fingerprinta(PMF)of proteins were indentified using MALDI-TOF-MS,and PMF data were retrieved by Profound and MASCOT. Comprehensive analysis of gel spectrum and mass spectrum data were used to select proteins constituting the oocyte maturation-related proteins in human cumulus cells.4. Bioinformatics analysis on differential proteinsVisited the bioinformatics websites:http://www.expasy.ch/sprot/, http://www. geneontology.org/, http://gather.genome.duke.edu/for querying the subcellular localization, biological process, molecular function and protein clustering of the differential expressed proteins, and analysed the significance of differences in protein expression by accessing to relevant literature in PubMed。Results1. After protein extraction, purification and quantification, protein concentrations of cumulus cells from Mature group and Immature group were 4.25mg/ml,3.82 mg /ml respectively.2. Difference gel electrophoresis patterns of cumulus cells differential proteome between two groups was aquired, and 25 different express protein spots was found by differential software analysis.3. Analysed by MALDI-TOF-MS and retrieved by Profound locally and Mascot on line separately,18 differential expressed proteins were identified with 8 up-and 10 down-regulated at mature group versus immature group.4. The proteins could be divided into five categories by bioinformatics analysis: enzymes involved in energy and substance metabolism, proteins involved in material transport and balance, proteins involved in genetic information storage and processing, proteins involved in cytoskeleton and cell division, and unclassified proteins.Summary1. The methodology of 2D-DIGE and MALDI-TOF-MS for proteomics analysis of human cumulus cells was established, and the 2D-DIGE protein profile of cumulus cells surrounding oocyte of different maturity was aquired successfully. 2. Analysed by MALDI-TOF-MS and retrieved by Profound locally and Mascot on line separately,18 differential expressed proteins were identified with 8 up-and 10 down-regulated at cumulus cells surrounding mature ooctes versus immature oocytes.3. These proteins could be divided into five categories by bioinformatics analysis: enzymes involved in energy and substance metabolism, proteins involved in material transport and balance, proteins involved in genetic information storage and processing, proteins involved in cytoskeleton and cell division, and unclassified proteins.They may be closely correlated to the active energy metabolism, transport, cell adhesion and movement, proliferation, division and other important biological processes in the cumulus cells during oocyte maturation.Chapter 2:Expression of Annexin A5 in human cumulus cellsObjectiveThe differential expressed protein identified by proteomics techniques may have important biological significance. But it was necessary to detect their expression to ensure its credibility and certainty of further research. Among the 18 differential expressed proteins been identified in Chapter one, Annexin A5 was one of the most highest reliable proteins which Total Ion C.I% is 100% in MELDI-TOF-MS, and it was 1.58 fold up-regulated at mature group versus immature group.In this chapter, Western Blot and Immunocytochemistry were performed to detect the different expression of Annexin A5 in cumulus cells surrounding oocyte of different maturity, and to analze the role of Annexin A5 in oocyte maturation.Methods1. Samples:The 62 subjects recruited in this study were undergoing ICSI at Nanfang Hospital Reproductive Center from February to May in 2010. Case inclusion criteria, cumulus cell collection and grouping were same to those in Chapter one.2. The expression of Annexin A5 protein in the cumulus cell was detected by Western Blot for semi-quantitative analysis..3. The expression of Annexin A5 protein in the cumulus cell was detected by immunocytochemistry for the subcellular localization and semi-quantitative analysis.4. SPSS 13.0 statistical package was used for data analysis. Measurement data was expressed by mean±standard deviation (x±s) and analyzed by using two independent samples t-test, Count data was analyzed by using Mann-Whitney test, P<0.05 was considered statistically significant.Results1. Western Blot:The expression of Annexin A5 protein were significantly stronger in the cumulus cell from Mature group than that from Immature group (t=4.64, P=0.010),which was consistant with the result from 2D-DIGE.2. Immunocytochemistry:Positive staining was observed at intracytoplasm and membrane of cumulus cells from the two groups. Analyzed by using Mann-Whitney test, Positive staining in the cumulus cell from Mature group were significantly higher than that from Immature group (Z=2.138, P=0.033)SummaryBy Westing Blot and immunocytochemistry, it is verified that the expression of Annexin A5 was up-regulated in the cumulus cells during oocyte maturation. This change may be closely correlated to the preovulatory LH peak, and related to the following cumulus expansion, the active energy metabolism, proliferation, extracellular matrix adhesion, Calcium oscillation and other important biological processes in the cumulus cells during oocyte maturation. Annexin A5 is probably a potential oocyte maturation-related protein in human cumulus cells.Chapter 3:Expression of CRBP-1 in human cumulus cellsObjectiveCRBP-1 was another one of the most highest reliable proteins amnong the 18 differential expressed proteins been identified in Chapter one, which Total Ion C.I% is 100% in MELDI-TOF-MS, and it was 1.73 fold down-regulated at mature group versus immature group. CRBP-1 was responsible for intracellular retinol transport in target cell, thus playing an important role in cellular uptake, transport, metabolism of retinol, and RBP-4 was responsible for retinol transport in blood and other body fluid.In this chapter, Western blot and immunocytochemistry were performed to detect the different expression of CRBP-1 in cumulus cells surrounding oocyte of different maturity, and ELASA were performed to detect the level of RBP-4 in follicular fluid of mature and immature human follicles, and to analyze the role of CRBP-1 and retinoids in oocyte maturation.Methods1. Samples:cumulus cell collection and grouping were same to those in Chapter two. Follicle fluids were from 58 subjects undergoing ICSI at Nanfang Hospital Reproductive Center from February to May in 2010. Case inclusion criteria were same to those in Chapter one. Follicle fluids were devided into two groups: Mature group and Immature group, according to the oocyte maturity.2. The expression of CRBP-1 protein in the cumulus cell was detected by Western Blot for semi-quantitative analysis.3. The expression of CRBP-1 protein in the cumulus cell was detected by immunocytochemistry for the subcellular localization and semi-quantitative analysis.4. The level of RBP-4 in the follicle fluids of mature and immature follicle was detected by ELISA.5. SPSS 13.0 statistical package was used for data analysis. Measurement data was expressed by mean±standard deviation (x±s) and analyzed by using two independent samples t-test, Count data was analyzed by using Mann-Whitney test, P<0.05 was considered statistically significant.Results1. Western Blot:The expression of Annexin A5 protein were significantly lower in the cumulus cell from Mature group than that from Immature group (t=10.760, P<0.001),which was consistant with the result from 2D-DIGE. 2. Immunocytochemistry:Positive staining was observed at intracytoplasm of cumulus cells from the two groups. Analyzed by using Mann-Whitney test, Positive staining in the cumulus cell from Mature group were significantly lower than that from Immature group (Z=1.977, P=0.048)3. ELISA:The RBP-4 level in mature follicle fluids and immature follicle fluids were 49.90±7.19μg/ml,38.48±9.46μg/ml respectively. Analyzed by using two independent samples t-test, the RBP-4 level in mature follicle fluids was significantly higher than that in immature follicle fluids (t=5.194, P<0.001)Summary1. By Westing Blot and immunocytochemistry, it is verified that the expression of CRBP-1 was down-regulated in the cumulus cells during oocyte maturation.2. CRBP-1 and RBP-4 were respectively responsible for the intracellular and extracellular retinol binding and transport. During the follicular development and oocyte maturation, the decreased CRBP-1 in the cumulus cells and the increased RBP-4 in follicular fluids may reflect decreased retinol storage and usage in cumulus cells and increased transport into the follicular fluids, the result of which was increased retinol uptake and used by oocyte. CRBP-1 is probably a potential oocyte maturation-related protein in human cumulus cells.conclusions1. The 2D-DIGE protein profile of cumulus cells surrounding oocyte of different maturity was aquired successfully, and 18 differential expressed proteins were identified with 8 up-and 10 down-regulated at cumulus cells surrounding mature oocyte versus immature oocyte.2. These differential expressed proteins could be divided into five categories by bioinformatics analysis:enzymes involved in energy and substance metabolism, proteins involved in material transport and balance, proteins involved in genetic information storage and processing, proteins involved in cytoskeleton and cell division, and unclassified proteins.They may be closely correlated to the active energy metabolism, transport, cell adhesion and movement, proliferation, division and other important biological processes in the cumulus cells during oocyte maturation.3. By Westing Blot and immunocytochemistry, it is verified that the expression of Annexin A5 was up-regulated in the cumulus cells during oocyte maturation. This change may be closely correlated to the preovulatory LH peak, and related to the following cumulus expansion, the active energy metabolism, proliferation, extracellular matrix adhesion, Calcium oscillation and other important biological processes in the cumulus cells during oocyte maturation. Annexin A5 is probably a potential oocyte maturation-related protein in human cumulus cells.3. By Westing Blot and immunocytochemistry, it is verified that the expression of CRBP-1 was down-regulated in the cumulus cells during oocyte maturation. During the follicular development and oocyte maturation, the decreased CRBP-1 in the cumulus cells and the increased RBP-4 in follicular fluids may reflect decreased retinol storage and usage in cumulus cells and increased transport into the follicular fluids, the result of which was increased retinol uptake and used by oocyte. CRBP-1 is probably a potential oocyte maturation-related protein in human cumulus cells. |
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