Yaakov Bentov1,2,3, Andrea Jurisicova4,5,6, Shlomit Kenigsberg7,6, Robert F Casper4,8,5,6. 1. Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada. ybentov@tcart.ca. 2. Toronto Center for Assisted Reproductive Technologies, Toronto, Canada. ybentov@tcart.ca. 3. Department of Obstetrics and Gynecology, University of Toronto, Toronto, Canada. ybentov@tcart.ca. 4. Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada. 5. Department of Physiology, University of Toronto, Toronto, Canada. 6. Department of Obstetrics and Gynecology, University of Toronto, Toronto, Canada. 7. CReATe Fertility Centre, Toronto, Canada. 8. Toronto Center for Assisted Reproductive Technologies, Toronto, Canada.
Abstract
PURPOSE: The purpose of the study was to establish the mechanism by which the estrogen concentration difference between the follicular fluid and the serum is maintained. METHODS: We used dialysis membrane with a pore size of <3 KD to characterize the estrogen-binding capacity of the follicular fluid. We performed PCR, western blot, and ELISA on luteinized granulosa cells to determine if sex hormone-binding globulin (SHBG) is produced by granulosa cells, and finally we used affinity columns and mass spectrometry to identify the estrogen-binding protein in the follicular fluid. RESULTS: We found that a significant estrogen concentration difference is maintained in a cell-free system and is lost with proteolysis of the follicular fluid proteins. Luteinized granulosa cells are likely not a source of SHBG, as we were not able to detect expression of SHBG in these cells. Perlecan was the most highly enriched follicular fluid protein in the affinity columns. CONCLUSIONS: We were able to identify perlecan as the most likely candidate for the major estrogen-binding protein in the follicular fluid.
PURPOSE: The purpose of the study was to establish the mechanism by which the estrogen concentration difference between the follicular fluid and the serum is maintained. METHODS: We used dialysis membrane with a pore size of <3 KD to characterize the estrogen-binding capacity of the follicular fluid. We performed PCR, western blot, and ELISA on luteinized granulosa cells to determine if sex hormone-binding globulin (SHBG) is produced by granulosa cells, and finally we used affinity columns and mass spectrometry to identify the estrogen-binding protein in the follicular fluid. RESULTS: We found that a significant estrogen concentration difference is maintained in a cell-free system and is lost with proteolysis of the follicular fluid proteins. Luteinized granulosa cells are likely not a source of SHBG, as we were not able to detect expression of SHBG in these cells. Perlecan was the most highly enriched follicular fluid protein in the affinity columns. CONCLUSIONS: We were able to identify perlecan as the most likely candidate for the major estrogen-binding protein in the follicular fluid.
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