Noga Chlamtac2, Zev Rosenwaks3, Leonid Poretsky1, Donna Seto-Young1 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

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 Muscle strength, def associated with increased fall Antiproliferative and induces differentiation used in psoriasis, induces apotosis Expressed on monocytes and macrophage and induces immunomodulator function, enhances rpoductuoion of cathelicidine a peptide capable of destroying infecious agents Holick MF 2007 N Engl J Med 357:266-281, Ahonen MH et al 2000 Int J Cancer 86:40-6

Vitamin D metabolism Ultraviolet B rays Dehydrocholestrol to previtamin D3 Excess vitamin D3 or previtamin D3 is destroyed by sunlight

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 Steroid hormone receptor family VDR forms heterodimer with retinoid X receptor which binds to DNA response element area and leads to gene In rat VDR is shown in males in seminiferous tubules, sertoli cells, spermatozoa In female in ovary, ovarian follicles specially granulosa cells, stroma and theca cells, corpus luteum 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,

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 Kinuta and colouges compared VDR nulll mice with heterozygous VDR null mice and wild type mice and showed that VDR null mice had lower estrogen levels and elevated FSH and LH levels, they were associated with imparied folliculogenesis, uterine hypoplasia which improved on estrogen supplementation 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

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 Kinuta and colouges compared VDR nulll mice with heterozygous VDR null mice and wild type mice and showed that VDR null mice had lower estrogen levels and elevated FSH and LH levels, they were associated with imparied folliculogenesis, uterine hypoplasia which improved on estrogen supplementation 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

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

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 203 bp 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)

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).

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)

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

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

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

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