Role of Vitamin D Receptor in Human Ovarian Cells and Steroidogenesis

1. Vitamin D Receptor (VDR) is Expressed in Human Ovarian Cells, likely Mediating the Effects of Vitamin D on Steroidogenesis Grishma Parikh1, Miroslava Varadinova1,Takako Araki1, Michael Goldman1, Noga Chlamtac2, Zev Rosenwaks3, Leonid Poretsky1, Donna Seto-Young1 1Division of Endocrinology & Friedman Diabetes Institute, Department of Medicine, Beth -- Israel Medical Center & Albert Einstein College of Medicine, New York, NY 10003 2Department of Medicine, New York University School of Medicine, New York, NY, 10016 3Center for Reproductive Medicine & Infertility, Weill Medical College of Cornell University, New York, NY 10021

2. Introduction • 1, 25-(OH)2D3 is active metabolite of vitamin D. Its primary role is to regulate calcium and phosphorus homeostasis and bone metabolism • Recently, many other actions of vitamin D have been identified. These include: • role in muscle physiology • immuno-modulator • anti-proliferative compound in a wide variety of cells including ovarian cancer cell lines Holick MF 2007 N Engl J Med 357:266-281, Ahonen MH et al 2000 Int J Cancer 86:40-6

3. Vitamin D metabolism

4. Introduction ( Cont.) • Effects of vitamin D are mediated through vitamin D receptor (VDR) - a member of nuclear receptor superfamily of ligand-activated transcription factors • VDR is expressed widely in human tissues including colon, muscle, brain, ovary, heart, lymphocytes, mono-nuclear cells • VDR is expressed in animal ovarian cells and appears to be necessary for ovarian function in animals • Vitamin D deficiency in female rats is associated with reduced fertility Agic A 2007 Reproductive Sciences 14:486-497, Holick MF 2007 N Engl J Med 357:266-281 Yoshizawa T Nat Genet 1997 16:391, Johnson JA Histochem Cell Biol 1996 105:7-15,

5. Introduction (Cont.) • VDR-null mutant mice have gonadal insufficiency. These mice also exhibit impaired folliculogenesis and uterine hypoplasia in females; reduced sperm motility and sperm count are observed in males • Calcitriol stimulates estradiol and progesterone production in trophoblast cells cultured from human placenta • Abnormalities of Vitamin D and calcium homeostasis as possible pathologic mechanism of PCOS Dukoh S Endocrinology 1983 112:200-6, Kinuta K 1999 Endocrinology 141:1317-1324, Barrera D 2007 Journal of Steroid Biochemistry & Molecular Biology 103:529–532, Thys-Jacobs S Steroids 1999 64:430–435

6. Introduction (Cont.) • VDR-null mutant mice have gonadal insufficiency. These mice also exhibit impaired folliculogenesis and uterine hypoplasia in females; reduced sperm motility and sperm count are observed in males • Calcitriol stimulates estradiol and progesterone production in trophoblast cells cultured from human placenta • Abnormalities of Vitamin D and calcium homeostasis as possible pathologic mechanism of PCOS • Role of vitamin D, if any, in human ovarian function and, in particular, in steroid hormone synthesis is not known Dukoh S Endocrinology 1983 112:200-6, Kinuta K 1999 Endocrinology 141:1317-1324, Barrera D 2007 Journal of Steroid Biochemistry & Molecular Biology 103:529–532, Thys-Jacobs S Steroids 1999 64:430–435

7. Human granulosa cells obtained duringin vitro fertilization Human ovarian tissue obtained duringoophorectomy Ovum retrieval during IVF Granulosa Cells Blood Cells Oocytes Tissue fragments Removal of oocytes for IVF Remaining separated by Percoll gradient Cut into 0.5x0.5 cm Granulosa Cells Mixed ovarian cell culture (granulosa, theca and stromal) Blood Cells 0.5x105 cells/ well in M199 tissue culture medium supplemented with 10% FBS. discard A A C B C B D D Incubated at 37°C, 5% CO2, 90% humidity Cell Cultures

8. Vitamin D Receptor mRNA expression in human ovarian cells by RT-PCR 1,25 (OH)2 vitamin D3 pM 0 50 100 150 nM 0 50 100 150 203 bp 203bp of VDR mRNA was detected in both human mixed ovarian cell culture and purified granulosa cell culture confirming the presence of VDR in human ovary and expression was not affected by exposure to 1, 25 (OH)2 D3 (0-150 pM or 0-150 nM)

9. Effect of 1,25-(OH)2 Vitamin D3 on steroid hormone production in mixed ovarian cell cultures 1, 25-(OH)2 D3 effect on steroid hormone production in mixed human ovarian and granulosa cell cultures. Steroid hormone production was assessed in the absence or presence of 1, 25-(OH)2 D3. Progesterone production in the mixed ovarian cells, , p<0.001 (n=13); testosterone production in the mixed ovarian cells, , p<NS (n=12); estradiol production in granulosa cells, , p<0.05 (n=5); estrone production in the granulosa cells, , p<0.002 (n=8).

10. Synergistic effect of 1,25-(OH)2 vitamin D3 with insulin on estradiol production in purified granulosa cell cultures Insulin alone has no significant effect on estradiol production in granulosa cell culture but addition of 1,25-(OH)2 vitamin D3 augments insulin induced estradiol production by up to 90% (p<0.006)

11. Effect of 1, 25-(OH)2 D3 on IGFBP-1 production 1, 25-(OH)2 D3 effect on IGFBP-1 production in human granulosa cells, , p<0.001 (n=7) compared to control [in the absence of 1, 25-(OH)2 D3]. Synergistic effect of 25-(OH)2 D3 with insulin on IGFBP-1 production in purified granulosa cell culture, y, p<0.009 (n=7) compared with the presence of insulin the absence of 25-(OH)2 D3 ; in the absence of (0 ng/ml insulin) or presence of (1-103 ng/ml) insulin, data are normalized to the baseline IGFBP-1 production of each experimental group

12. Conclusions • This report confirms presence of VDR in human ovarian cells and suggests that Vitamin D may have a physiological role in the human ovary • 1, 25-(OH)2D3 stimulated steroid hormone production including progesterone, estradiol and estrone. Its effect on testosterone production was not significant. It may play a role in regulation of human ovarian steroidogenesis • Addition of 1, 25-(OH)2D3 enhances insulin-induced estradiol production, possible mechanism being increased insulin sensitivity in ovarian tissue

13. Significance These findings likely suggest role of vitamin D in ovarian regulation and importance of detecting and correcting Vitamin D deficiency especially in young women

14. Acknowledgements We are thankful for the support from: • Gerald J. and Dorothy R. Friedman Foundation • Chinese American Medical Association & Chinese American Independent Practice Association