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

Grishma Parikh1, Takako Araki1, Michael Goldman1, 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

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

Vitamin D metabolism

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,

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

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

Cell Cultures

Ovum retrieval during IVF

Human granulosa cells obtainedduring in vitro fertilization

Granulosa Cells Blood Cells Oocytes

Removal of oocytes for IVF Remaining separated by Percoll gradient

ABC

D

discard

Blood CellsGranulosa Cells

0.5x105 cells/ well in M199 tissue culture medium supplemented with 10% FBS.

Human ovarian tissue obtainedduring oophorectomy

Tissue fragments

Cut into 0.5x0.5 cm

Mixed ovarian cell culture(granulosa, theca and stromal)

ABC

DIncubated at 37°C, 5% CO2, 90% humidity

Vitamin D Receptor mRNA expression in human ovarian cells by RT-PCR

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

341 bp

258 bp

203 bp

Mixed ovarian cell culture Granulosa cell culture

Effect of 1,25-(OH)2 Vitamin D3 on steroid hormone production in mixed ovarian cell cultures

Progesterone Testosterone60

70

80

90

100

110

120

130

control50-150nM Vitamin Dp<0.001

N=9

N=11

Hor

mon

e co

ncen

tratio

n in

tiss

ue c

ultu

re m

ediu

m(%

con

trol ±

SE

M)

1, 25-(OH)2D3 stimulated progesterone production in mixed ovarian cell culture by 15% (p<0.001) compared to control and had no effect on testosterone production

Effect of 1,25-(OH)2 vitamin D3 on steroid hormone production in purified granulosa cells

Estradiol Estrone70

80

90

100

110

120

130

control50-150nM Vitamin D

p<0.05N=6

p<0.002N=8

Hor

mon

e co

ncen

tratio

n in

tiss

ue c

ultu

re m

ediu

m(%

con

trol ±

SE

M)

1, 25-(OH)2D3 stimulated estradiol and estrone production in granulosa cell culture by 6% (p<0.05) and 21% (p<0.002) respectively compared to control

Insulin (ng/ml)

0 10 100 100080

100

120

140

160

180

200

220

Control100nM Vit D

Est

radi

ol c

once

ntra

tion

in ti

ssue

cul

ture

med

ium

(% c

ontro

l ± S

EM

) P<0.006

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)

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 suggest role of vitamin D in ovarian regulation and

importance of detecting and correcting Vitamin D deficiency, especially in reproductive age

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