Abstract
Introduction
Ovarian follicles require a robust vascular supply to grow and ovulate. Dysfunction of ovarian vasculature impairs follicle growth and ovulation. The ovarian vasculature is in close proximity to theca cells, which produce androgens. Women with PCOS have altered macrovascular and microvascular function as well as elevated androgens. We hypothesize that the response of ovarian microvascular endothelial cells to androgens may be impaired in PCOS.
Methods
Ovarian microvascular endothelial cells from healthy oocyte donors (HOMECs) and PCOS patients (PCOMECs) were isolated from discarded follicular aspirates. HOMEC and PCOMEC expression of classical androgen receptor AR and plasma membrane androgen receptor SLC39A9 mRNA was determined via qPCR, and protein was assessed through immunostaining of fixed cells and Western blot of cell lysate. HOMEC and PCOMEC response to androgens via AR was determined using gradient-driven migration and live cell tracking assays. Response to androgens via SLC39A9 was determined by zinc influx using Zinquin ethyl ester and proliferation via BrdU incorporation.
Results
HOMECs and PCOMECs expressed mRNA and protein for AR and SLC39A9. PCOMEC AR mRNA expression was 138% greater than that of HOMECs (p 0.05), with no difference in SLC39A9 mRNA between HOMECs and PCOMECs. HOMEC migration was increased by 62% in the presence of testosterone (T; p 0.05), but not the membrane impermeable bovine serum albumin-conjugated T (BSA-T), whereas PCOMEC migration was not altered by any stimulus. siRNA knockdown of AR blocked T-stimulated migration in HOMECs (p 0.05) while siRNA knockdown of SLC39A9 had no impact on basal or T-stimulated migration. PCOMEC migration in the presence of T was not affected by siRNA knockdown of SLC39A9 or AR. Live cell tracking showed that T increased speed and distance traveled by migrating HOMECs, while T had no impact on migrating PCOMECs. T stimulated zinc influx in a dose dependent manner in HOMECs (93% increase, p 0.05), but not in PCOMECs. Additionally, T, BSA-T, and the SLC39A9-selective agonist (-)-epicatechin each increased proliferation in HOMECs by 300-500% (p 0.05), with no change in PCOMEC proliferation. siRNA knockdown of SLC39A9 blocked T-stimulated proliferation in HOMECs (p 0.05) while siRNA knockdown of AR had no impact on basal or T-stimulated proliferation. PCOMEC proliferation in the presence of T was not affected by siRNA knockdown of SLC39A9 or AR. These findings indicate that androgens stimulate migration via AR and increase zinc influx and proliferation via SLC39A9 in HOMECs, but not in PCOMECs.
Conclusion
Overall, androgens act via both SLC39A9 and AR at HOMECs to stimulate proliferation and migration, which are key processes in angiogenesis. Importantly, although PCOMECs express SLC39A9 and AR, androgens do not stimulate angiogenic responses via SLC39A9 or AR in PCOMECs. Local androgen action may be central to ovarian vascular remodeling and ovulatory angiogenesis in healthy ovarian function. Absent or aberrant function of androgen signaling in PCOMECs may contribute to ovarian vascular dysfunction and may ultimately play an important role in the pathology of PCOS.