OBJECTIVE: To examine the differences in specific protein expression between mural and cumulus granulosa cells following 24-hour in vitro culture. DESIGN: Laboratory study. SETTING: University Hospital. INTERVENTION(S): Human granulosa cells were collected at the time of egg collection during assisted reproduction. Cumulus cells associated with the oocyte were separated from mural cells from the periphery of the follicle before in vitro culture for 24 hours. Cells were then lysed and subjected to two-dimensional gel electrophoresis. MAIN OUTCOME MEASURE(S): Given that cumulus (cGC) and mural granulosa cells (mGC) differentiate from a single layer, it is likely that phenotypic differences between them may reflect specific molecular processes and structural adaptations. Computer-assisted analysis using dedicated software enabled the presence, absence, or relative volume of each individual protein spot to be estimated. Differentially expressed spots were identified using tandem mass spectrometry. RESULT(S): The mean number of separate protein spots detected in mGC gels was 1,105 +/- 146, and in cGC it was 887 +/- 236, although there was no statistically significant difference between the two. Five enzymes of the glycolytic pathway were never expressed in cGC after 24 hours in vitro; these were triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase 1, and two isoforms of alpha enolase. These are the first data collected in humans consistent with a recent demonstration that isolated murine cGC cultured in vitro exhibit decreased expression of mRNA encoding glycolytic enzymes, and support the suggestion that some factor or factors secreted by the oocyte may be responsible for the maintenance of glycolysis in the adjacent cGC.
OBJECTIVE: To examine the differences in specific protein expression between mural and cumulus granulosa cells following 24-hour in vitro culture. DESIGN: Laboratory study. SETTING: University Hospital. INTERVENTION(S): Human granulosa cells were collected at the time of egg collection during assisted reproduction. Cumulus cells associated with the oocyte were separated from mural cells from the periphery of the follicle before in vitro culture for 24 hours. Cells were then lysed and subjected to two-dimensional gel electrophoresis. MAIN OUTCOME MEASURE(S): Given that cumulus (cGC) and mural granulosa cells (mGC) differentiate from a single layer, it is likely that phenotypic differences between them may reflect specific molecular processes and structural adaptations. Computer-assisted analysis using dedicated software enabled the presence, absence, or relative volume of each individual protein spot to be estimated. Differentially expressed spots were identified using tandem mass spectrometry. RESULT(S): The mean number of separate protein spots detected in mGC gels was 1,105 +/- 146, and in cGC it was 887 +/- 236, although there was no statistically significant difference between the two. Five enzymes of the glycolytic pathway were never expressed in cGC after 24 hours in vitro; these were triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase 1, and two isoforms of alpha enolase. These are the first data collected in humans consistent with a recent demonstration that isolated murine cGC cultured in vitro exhibit decreased expression of mRNA encoding glycolytic enzymes, and support the suggestion that some factor or factors secreted by the oocyte may be responsible for the maintenance of glycolysis in the adjacent cGC.