What maintains the high intra-follicular estradiol concentration in pre-ovulatory follicles?

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.

Keywords: Estrogen binding; Follicular fluid; Perlecan; SHBG.

Fig. 1

SHBG is not expressed in granulosa cells. a RT-PCR for multiple segments of the sex hormone-binding globulin (SHBG) mRNA in human granulosa cells, liver, and testis. Results show no expression of any part of the SHBG mRNA in granulosa cells. The top panel shows SHBG exons 1–6, second panel shows SHBG exons 6–8, and the third panel shows SHBG exons 3–8 with the human testis and liver but not with two pools of GC. The bottom panel shows human GAPDH as positive control. hTestis human testis, hLiver human liver, GC granulosa cells, NTC no template control. b Real-time PCR for multiple segments of SHBG mRNA in the human granulosa cells, liver, and testis. Results show no expression of any part of the SHBG mRNA in granulosa cells. c Western blot analysis for the SHBG protein in human granulosa cells as well as ovarian carcinoma cell line and follicular fluid and actin as a positive control. Results show a clear detection of SHBG in follicular fluid but none in granulosa cells and ovarian carcinoma cell line

Fig. 2

Mass spectrometry analysis of avidin biotin fractions. a This figure illustrates the concentration of the proteins in the flow-through (the native follicular fluid that passed through the column before any washes were made) on the x-axis and the first biotin wash on the y-axis. Albumin was the most abundant protein in the flow-through fraction that contains the proteins that had passed the avidin column without binding. At the other end the HSPG2 protein that could not be detected in the flow-through but had the highest concentration in the biotin wash which is enriched with specifically bound proteins. b This Venn diagram represents the distribution of the detected proteins in the different fractions. In yellow is the group of proteins that were detected in the follicular fluid but not in the flow-through and were released from the column with the biotin wash. The bar graph represents the relative concentration of the 11 proteins exhibiting specific binding to the avidin biotin-bound estradiol. HSPG2, also known as perlecan, had the highest enrichment following the biotin wash