Transcript Slide 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
3Center
of Medicine, New York University School of Medicine, New York, NY, 10016
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
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
Human granulosa cells obtained
during in vitro fertilization
Human ovarian tissue obtained
during oophorectomy
Ovum retrieval during IVF
Granulosa Cells
Blood Cells
Oocytes
Tissue fragments
Removal of oocytes for IVF
Remaining separated by Percoll gradient
Blood Cells
Granulosa Cells
Mixed ovarian cell culture
(granulosa, theca and stromal)
0.5x105 cells/ well in M199 tissue culture medium
supplemented with 10% FBS.
discard
C
A
D
Cut into 0.5x0.5 cm
B
C
Incubated at 37°C, 5% CO2, 90% humidity
A
D
B
Vitamin D Receptor mRNA expression in human ovarian cells
by RT-PCR
1,25 (OH)2 vitamin D3
pM
nM
0
50
100
150
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)
Hormone concentration in tissue culture medium
(% control ± SEM)
Effect of 1,25-(OH)2 Vitamin D3 on steroid
hormone production in mixed ovarian cell
cultures
130
120
control
50-150 pM, 50-150 nM
Vitamin D
0.001)
0.002)
0.05)
110
(NS)
100
90
80
Progesterone
Testosterone
Estradiol
Estrone
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).
Estradiol concentration in tissue culture medium
(% control ± SEM)
Synergistic effect of 1,25-(OH)2 vitamin D3 with insulin on
estradiol production in purified granulosa cell cultures
180
Control
50-150 pM; 100nM Vit D
160
140
120
100
80
0
10
100
1000
Insulin (ng/ml)
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
140
control
50-150 pM, 50-150 nM
Vitamin D
(0.001)
control
50-150 pM, 50-150 nM
Vitamin D
100
120
100
80
60
40
20
0
Control
IGFBP-1 production (% control ± SEM)
IGFBP-1 production (% control ± SEM)
160
Vitamin D
80
60
40
(0.009)
20
0
10
100
1000
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