Presentations

Abstract

Introduction:

Follicular androgens are important for oocyte health and fertilization. The classical nuclear androgen receptor (AR) is a transcription factor expressed in the cells of the ovarian follicle. Androgen actions are also mediated via the plasma membrane androgen receptor SLC39A9. First characterized as a zinc transporter, studies in fish ovary demonstrated that androgens bind to SLC39A9, increase intracellular zinc, and stimulate granulosa cell apoptosis. Intracellular zinc in ovarian cells is known to impact ovulation, but expression and function of SLC39A9 have not been investigated in the mammalian ovary to date. We hypothesize that SLC39A9 is expressed in the key cell types of the monkey ovulatory follicle and that androgen activation of SLC39A9 increases intracellular zinc to mediate ovulatory changes in ovarian cells.

Methods:

Adult, female cynomolgus macaques experienced ovarian stimulation. Monkey ovaries were harvested across the 40-hour ovulatory window at 0, 12, 24, and 36 hours after hCG stimulation, and ovarian cells were isolated from follicular aspirates (granulosa, vascular endothelial cells) or follicle theca layers (theca cells). AR and SLC39A9 mRNA was quantitated via qPCR. Ovary cells and tissue sections were immunostained for AR and SLC39A9. Effects of androgens on cultured ovarian cells were assessed by in vitro treatment with testosterone (T; 2.5-50 nM), BSA-conjugated testosterone (BSA-T; 10-25 nM), or androstenedione (A4; 100-300 nM). Intracellular zinc levels were assessed via incubation of cells with Zinquin ethyl ester and fluorescence detection via microplate reader. Proliferation was assessed via Ki-67 immunodetection. Vascular endothelial cell migration through a permeable membrane in vitro was assessed by counting migrated cells after hematoxylin and eosin staining of the membrane.

Results:

AR and SLC39A9 mRNA was present in granulosa, theca, and vascular endothelial cells. Immunostaining for AR and SLC39A9 demonstrated protein expression in all key cell types of the follicle across the ovulatory window. Treatment with T, BSA-T, and A4 stimulated zinc influx in granulosa, theca, and vascular endothelial cells. Proliferation was increased after 48 hour treatment with T, BSA-T, and A4 in vascular endothelial cells but not in granulosa or theca cells. Vascular endothelial cell migration was increased after 24-hour treatment with T but not with BSA-T or A4.

Conclusion:

These results support the hypothesis that the novel membrane androgen receptor SLC39A9 is expressed and functional in cells of the monkey ovulatory follicle. Ovarian granulosa, theca, and vascular endothelial cells express AR and SLC39A9 mRNA and protein. T is the highest affinity ligand for SLC39A9. BSA-T is membrane impermeable and thus only able to interact with plasma membrane receptors, while A4 is the predominant androgen in the primate ovulatory follicle in vivo. Increase of intracellular zinc in response to all three androgens supports the conclusion that SLC39A9 activation via androgen induces zinc influx in ovarian granulosa, theca, and vascular endothelial cells. Activation in response to the A4 concentration present in the ovulatory follicle supports the concept that SLC39A9 could be activated during ovulation. Increased vascular endothelial cell proliferation in response to all three androgens supports the involvement of SLC39A9 in ovulatory angiogenesis. However, increased migration in response to T alone suggests that androgens stimulate vascular endothelial cell migration through nuclear AR. Activation of SLC39A9 but not nuclear AR by follicular A4 concentrations implies a higher affinity of A4 for SLC39A9, and thus suggests a pathway for A4 action through SLC39A9 in the ovulatory follicle. Future studies will further investigate SLC39A9 mediation of androgen action at granulosa and theca cells during ovulation. Supported by funding from EVMS and product donation from Organon, Jersey City, NJ.