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Department of Pediatrics, Okayama University Medical School (K.K., H.T., T.M., K.A., Y.S.), Okayama 700-8558; and Institute of Molecular and Cellular Biosciences (S.K.), University of Tokyo, Tokyo 113-0032, Japan
Address all correspondence and requests for reprints to: Dr. Hiroyuki Tanaka, Department of Pediatrics, Okayama University Medical School, 2–5-1 Shikata-cho, Okayama 700-8558, Japan. E-mail: hrtanaka{at}hospital.okayama-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß were normal in gonads in
these mice. Supplementation of estradiol normalized histological
abnormality in the male gonads as well as in the female. Calcium
supplementation increased aromatase activity and partially corrected
the hypogonadism. When the serum calcium concentration was kept in the
normal range by supplementation, the aromatase activity in the ovary
increased to 60% of the wild-type level, but LH and FSH levels were
still elevated. These results indicated that vitamin D is essential for
full gonadal function in both sexes. The action of vitamin D on
estrogen biosynthesis was partially explained by maintaining calcium
homeostasis; however, direct regulation of the expression of the
aromatase gene should not be neglected. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To clarify the pathophysiology of the disorder of gonads in the VDR null mutant mice, the activity of aromatase cytochrome P450 (P450arom), a key enzyme in estrogen biosynthesis, and the expression of the CYP19 gene encoding P450arom (8) were investigated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
VDR null mutant mice were generated by gene targeting as
described previously (7); the locus targeted for the disruption of the
VDR gene included exon 2, and the mutant locus contained the neomycin
resistance gene. Mice were weaned at 3 weeks of age and were then fed
ad libitum distilled water and a chow diet (MF, Oriental
Yeast, Tokyo, Japan; ingredients: 11.1 mg/g calcium, 8.3 mg/g
phosphorus, and 1.08 IU/g vitamin D3) or a high
calcium diet (9) (Clea Japan, Inc., Tokyo, Japan; ingredients: 20.0
mg/g calcium, 12.5 mg/g phosphorus, 1.08 IU/g vitamin
D3, and 200 mg/g lactose). The mice were
maintained under specific pathogen-free conditions with a 12-h light,
12-h dark cycle. They were bred as heterozygotes. The studies were
reviewed and approved by the institutional committee of animal care and
use of Okayama University Medical School.
The VDR genotypes were determined by analyzing genomic DNA obtained from each mouse at approximately 10 days after birth. Genomic DNA was extracted from tail clippings with the QIAamp tissue kit and was amplified by multiplex PCR using two sets of primers specific for the wild-type exon 2 of VDR gene and for the neomycin resistance gene, respectively. To this end, we recloned murine exon 2 of VDR gene and sequenced it. Novel primers with the sequences of 5'-CCT CCA TCC CTG TAA GAA GA-3' and 5'-CAA AGA ACT GCC ACC CAC TC-3' were prepared. Another set of primers (5'-TGA ATG AAC TGC AGG ACG AGG-3' and 5'-AAG GTG AGA TGA CAG GAG ATC-3') for detection of the neomycin resistance gene (10) was also prepared. The reaction mixture (50 µl) contained DNA template (4 µl), two of the primer sets (0.4 µM each), Taq polymerase (1 U), 5 µl of 10 x PCR buffer, MgCl2 (1.5 mM), and a deoxy-NTP mixture (0.2 mM). The amplification included an initial denaturation step at 94 C for 5 min, followed by 30 cycles of denaturation at 94 C (1 min), annealing at 55 C (1 min), and extension at 72 C (2 min), and an additional extension step of 72 C for 7 min. The amplified products were analyzed by 3% agarose gel electrophoresis and ethidium bromide staining. The expected sizes of the products were 130 bp for exon 2 of VDR and 150 bp for the neomycin resistance gene.
Determination of P450arom activity
Activities of P450arom in ovaries were determined from the
liberation of [3H]H2O
from [1ß-3H]androstenedione essentially
according to previously reported methods (11, 12). The ovaries were
homogenized with 10 times the volume of 10 mM potassium
phosphate buffer (pH 7.4). The reaction was carried out by incubating a
200-µl reaction mixture containing potassium phosphate buffer (pH
7.4), [1ß-3H]androstenedione (200
nM; 0.988 Ci), NADPH (10 mM), and 20 µl of
the homogenate (10–20 µg protein) at 37 C for 5–30 min and was
terminated by adding 100 µl 25% (wt/vol) trichloroacetic acid. To
remove unreacted [1ß-3H]androstenedione, the
reaction mixture was treated with 100 µl dextran-charcoal, then
centrifuged at 1200 x g for 5 min to obtain the
supernatant. To further remove
[1ß-3H]androstenedione, a 300-µl aliquot of
the supernatant was diluted with 700 µl water and extracted with 2.5
ml chloroform. The radioactivity of the aqueous layer containing
[3H]H2O was measured in a
liquid scintillation counter. A reaction mixture without the homogenate
was used as a blank. From linear plots of the amounts of the product
against reaction times, P450arom activities were determined in terms of
picomoles of [3H]H2O
liberated per min/mg protein. Protein contents were determined using a
bicinchoninic acid kit (Pierce Chemical Co., Rockford,
IL).
The method for determination of P450arom activities in the testis and epididymis was the same as that employed for the ovary samples, except for cofactors and incubation time, because of the lower activity of the aromatase. The activities of P450arom in microsomal fractions prepared from the testis or the epididymis of male mice (10 weeks old) were determined as previously described (13). A 200-µl reaction mixture containing 10 mM potassium phosphate buffer (pH 7.4), MgCl2 (85 mM), NADP+ (10 mM), NADPH (10 mM), glucose-6-phosphate (100 mM), [1ß-3H]androstenedione (200 nM; 0.988 Ci), glucose-6-phosphate dehydrogenase (2 U), and 20 µl of the microsomal fractions as the enzyme source was incubated at 37 C for 1 h. A sample for determination of radioactivity was prepared in the manner described above. P450arom activity was defined in terms of picomoles of [3H]H2O liberated per h/mg protein.
RT-PCR analysis for CYP19 gene expression and estrogen receptor
(ER) and ERß gene expression
Total RNA was extracted from the ovary and the testis by the
acid-guanidine phenol-chloroform method (14). A RT-PCR analysis was
carried out using 2 µg total RNA from the ovary and 5 µg total RNA
from the testis, which was reverse transcribed using a random primer
and Moloney murine leukemia virus reverse transcriptase in 25
µl, according to the manufacturer’s protocol. An aliquot of the RT
reaction was then used as the template for a subsequent PCR.
The complementary DNA (cDNA) for the mouse CYP19 gene was analyzed by PCR using a 50-µl reaction mixture containing cDNA template (2 µl from the ovary sample, 4 µl from the testis sample), specific primers of 5'-TGA GAG ACG TGG AGA CCT GA-3' and 5'-CAC CTG GAA TCG TCT CAA AA-3' (0.4 µM each), Taq polymerase (1 U), 5 µl 10 x PCR buffer, MgCl2 (1.75 mM), and a deoxy-NTP mixture (0.2 mM). The reaction procedure for the amplification was as follows: an initial denaturation step at 95 C for 5 min; 35 cycles of 95 C (1 min), 56 C (2 min), and 72 C (2 min); and an extension step at 72 C for 10 min. The products were analyzed by 2% agarose gel electrophoresis and ethidium bromide staining. The expected size of the products was 526 bp. Direct sequencing of the 526-bp PCR product revealed a corresponding sequence in P450arom messenger RNA (mRNA). The image of the UV-illuminated gels was stored in a digital form and analyzed by a computerized image analyzing system (ATTO densitograph, ATTO Corp., Tokyo, Japan).
The cDNA for ER and ERß genes were analyzed by previously
described methods (15, 16).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was coamplified to serve as an internal control. Each band was normalized to the value of GAPDH.
Competitive PCR analysis for quantitative CYP19 gene
expression
The cDNA for mouse CYP19 gene after the calcium supplementation
was analyzed by competitive PCR. The competitive sequence was
constructed by PCR using PCR MIMICS as a template according to the
manufacturer’s protocol. The competitive template used was 4, 4
x 10-1, 4 x 10-2,
4 x 10-3, 4 x
10-4 fM in each 50-µl
reaction mixture. The expected size of the products was 350 bp.
Serum chemistries
LH levels and FSH levels were measured using an enzyme-linked
immunosorbent assay for rat LH and FSH (Amersham Pharmacia Biotech, Aylesbury, UK). Estradiol levels were measured by RIA
(Diagnostic Products, Los Angels, CA) at Mistubishi BCL
(Tokyo, Japan). Calcium levels were measured using the
o-cresol phthalein complexion method (Wako, Osaka, Japan).
Phosphorous levels were measured using the
p-methylaminophenol method (Wako).
Sperm function
Sperm counts and motility were determined. Sperm were collected
from the epididymides of 11-week-old heterozygous and VDR null mutant
mice. Sperm suspensions were prepared by mincing the excised cauda
epididymides in 0.5 ml capacitation medium (17, 18). After allowing 15
min for sperm dispersion, particulate tissue was removed, and aliquots
of the epididymal suspension were diluted 1:5 in the medium. Sperm
counts and the estimated percentage of motile sperm were determined
visually by phase microscopy.
Histological analysis
Testes were removed from mice, preincubated with OCT compound
(Miles, Elkhart, IL), and then frozen with liquid nitrogen. Five-micron
sections, cut with a cryostat, were collected on
poly-L-lysine-coated slide glasses. The sections were
stained with methyl green.
Estrogen supplementation
17ß-Estradiol was given to VDR null mutant male mice at 5–10
weeks of age (10 ng/head·day) (19) by microosmotic pump (Alzet, Palo
Alto, CA). After 5 weeks of treatment with 17ß-estradiol, mice were
killed, and the histology of the testes, sperm function, aromatase
activity in the testes, the expression level of the CTP19 gene in the
testes, and serum levels of calcium, LH, and FSH were analyzed.
17ß-Estradiol was given to 7-week-old VDR null mutant female mice
(100 ng/head·day) (19) by ip injection for 7 days. After treatment
with 17ß-estradiol, mice were killed, and aromatase activity in the
ovaries was measured.
Statistical analysis
Values are given as the mean ± SEM.
Statistical analysis was performed using unpaired Student’s
t test and ANOVA, followed by Fisher’s protected least
significant difference. P < 0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. In the ovaries of the wild-type
(VDR+/+) mice, the enzyme activities at 4 and 7
weeks of age were 0.102 ± 0.018 (mean ± SEM;
n = 4) and 0.357 ± 0.048 (n = 4) pmol
[3H]H2O
liberated/min·mg protein, respectively. In the 7-week-old
heterozygous (VDR+/-) mice, the activity was
0.295 ± 0.035 (n = 4). No significant difference was found
between the VDR+/+ and
VDR+/- mice in P450arom activity. Samples from
the VDR+/+ and VDR+/-
female mice at 7 weeks of age were obtained at the time of estrus, as
evidenced by labial swelling and reddening of vaginal membranes. The
VDR null mutant (VDR-/-) female mice never
showed such a genital appearance. P450arom activity in the 7-week-old
VDR-/- mice was 0.087 ± 0.011 (n =
4). This value was 24.4% of that in the 7-week-old
VDR+/+ mice and was similar to that in the
4-week-old VDR+/+ mice (just after weaning).
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, P450arom activity in the testes of 10-week-old
VDR+/+ mice was 0.354 ± 0.020 pmol
[3H]H2O liberated/h·mg
protein (mean ± SEM; n = 4), which is
approximately 1/60th the value in the ovaries of 7-week-old
VDR+/+ female mice. P450arom activity in the
testes of 10-week-old VDR-/- mice was
0.207 ± 0.028 (n = 5) pmol
[3H]H2O liberated/h·mg
protein. This level was 58.5% of that in 10-week-old
VDR+/+ mice (P < 0.005). In the
epididymis, the level of P450arom activity in 10-week-old
VDR-/- mice was 0.225 ± 0.049, which was
34.6% of that in VDR+/+ mice (0.650 ±
0.118; n = 4; P < 0.01).
To evaluate the level of CYP19 gene expression, we applied a RT-PCR
procedure. The results presented in Fig. 2 indicated that the ovary and testis of
VDR-/- mice expressed the mRNA of the CYP19
gene, but the expression levels of the CYP19 gene were markedly
decreased. Data from scans of the PCR gels are provided in Table 1. The expression levels of CYP19 gene in
the ovary and testis of heterozygous (VDR+/-)
mice were similar to those in VDR+/+ mice (data
not shown). The PCR reactions without RT had no products. The results
of competitive PCR were consistent with those of this RT-PCR.
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, the sperm counts in
VDR-/- mice were half those in
VDR+/- mice. These results revealed that the
number of functional sperm was markedly decreased in
VDR-/- mice compared with
VDR+/- mice. The motility and sperm count in
VDR+/- mice were similar to those in wild-type
mice (18).
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gene knockout
mice (Fig. 3).
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, the testes of 10-week-old
VDR-/- mice showed dilated lumen of
seminiferous tubules, a thinner layer of epithelial cells, and
decreased spermatogenesis. The testes of 15-week-old
VDR-/- mice revealed rare spermatogenesis. A
more widely dilated lumen of seminiferous tubules and atrophy of the
seminiferous epithelium cells were observed (Fig. 4C).
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. The circulating
estradiol level in VDR null mutant female mice was significantly lower
than that in heterozygous mice. Serum levels of LH and FSH in VDR null
mutant mice are shown in Table 4. The LH
and FSH levels in 3-week-old VDR null mutant mice were the same as
those in VDR+/+ mice. The LH level was elevated
5- to 10-fold in both female and male VDR-/-
mice compared with that in VDR+/+ mice at 8 weeks
of age. The FSH level in VDR-/- mice was twice
that in VDR+/+ mice at 8 weeks of age. These
elevated levels of LH and FSH in VDR-/- mice
were sustained to 12 weeks of age. The estradiol, LH, and FSH levels
were measured in wild-type female mice that were in estrous, as
evidenced by labial swelling and reddening of vaginal membranes.
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. The sperm count and motility in the estrogen-treated
mice (n = 3) were increased to the same level in the heterozygous
mice [count, 47.7 ± 5.0 x 106/ml
vs. 26.3 ± 7.3 x 106/ml in
VDR-/- without treatment (P <
0.05); motility: 47.0 ± 3.6% vs. 6.0 ± 4.6% in
VDR-/- without treatment (P =
0.0001)]. The P450arom activity in the testes of
VDR-/- mice with estrogen treatment was
increased (0.328 ± 0.018 pmol
[3H]H2O liberated/h·mg
protein vs. 0.207 ± 0.028 in the
VDR-/- without treatment; n = 3;
P = 0.03). The expression level of the CYP19 gene in
the testis with estrogen supplementation was analyzed by competitive
PCR. The level in VDR-/- was 0.6 ± 0.2
attomoles (attmol; 10-18
M). After estrogen treatment, the expression
level was significantly increased to 16.7 ± 3.3 attomol
(P < 0.0001). This level was similar to the level in
VDR+/+ (23.3 ± 8.8 attomol; P = 0.613). The serum
LH level was decreased to 4.13 ± 0.20 ng/ml (n = 3), and the
serum FSH level was decreased to 208.7 ± 10.5 ng/ml (n = 3)
after the supplementation (P = 0.0003 and <0.0001
respectively, compared with VDR-/- male mice at
8 weeks of age). The serum calcium level was not increased (6.4 ±
0.1 vs. 8.30 ± 0.26 mg/dl in the
VDR+/+ mice; P = 0.0008). As in
the male mice, the P450arom activity in the ovary of
VDR-/- mice (n = 3) with estrogen
treatment was increased (0.293 ± 0.028 pmol
[3H]H2O
liberated/min·mg protein vs. 0.087 ± 0.011 in the
VDR-/- without treatment; P <
0.0001).
The expression levels of ER and ERß in the ovary and testis
of the VDR null mutant mice were the same as those in the wild-type
mice (Fig. 5). In the ovary, the ratios
of the expression level of ER/GAPDH in
VDR-/- and VDR+/+ were
0.877 ± 0.167 and 0.998 ± 0.078, respectively, and the
ratios of the expression level of ERß/GAPDH in
VDR-/- and VDR+/+ were
7.190 ± 0.622 and 8.647 ± 0.891, respectively. In the
testis, the ratios of the expression level of ER/GAPDH in
VDR-/- and VDR+/+ were
0.613 ± 0.029 and 0.642 ± 0.057, respectively, and the
ratios of the expression level of ERß/GAPDH were 0.968 ± 0.109
and 1.027 ± 0.084, respectively. The scanned data revealed that
there was no significant difference between
VDR-/- and VDR+/+ mice in
either ER expression.
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and Table 5. These results indicated that
the expression of the CYP19 gene was increased by calcium
supplementation in both ovary and testis. However, as with aromatase
activity, the expression level of the aromatase gene was not completely
recovered after normalization of serum calcium, being one fifth that of
the wild-type value in the ovary (P = 0.004) and one
fourth that of the wild-type value in the testis (P =
0.02). As shown in Table 3, the circulating estradiol level did not
increase after calcium supplementation. In the female the estradiol
level was 5.6 ± 2.6 pg/ml (n = 4; P = 0.03
compared with that in VDR+/- female mice at 8
weeks of age). The serum levels of LH and FSH were not corrected and
remained high despite the fact that the serum calcium level was normal
after calcium supplementation. In the female, the LH level was 5.9
± 0.2 ng/ml, and the FSH level was 385 ± 32 ng/ml (n = 3;
P < 0.0001 and 0.001, respectively, compared with that
in VDR+/+ female mice at 8 weeks of age). In male
mice, the LH level was 8.1 ± 0.3 ng/ml (n = 3;
P < 0.0001 compared with VDR+/+
male mice at 8 weeks of age), and the FSH level was 310 ± 16
ng/ml (n = 3; P < 0.0001 compared with
VDR+/+ male mice at 8 weeks of age). In male VDR
null mutant mice given calcium supplements, dilated lumens were
observed in certain seminiferous tubules (Fig. 4E). The sperm count was
increased to 44.0 ± 5.3 x 106/ml, but
was not significantly different compared with that in
VDR-/- animals without treatment
(P = 0.09). Sperm motility was significantly increased
to 39.3% compared with that in VDR-/- mice
without treatment (P = 0.0015).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In female VDR null mutant mice, uterine hypoplasia with impaired
folliculogenesis was observed, and estrogen supplementation increased
uterine weight (7). These results indicated that estrogen deficiency
caused impaired folliculogenesis and uterine hypoplasia in female VDR
null mutant mice. In male VDR null mutant mice in this study, a
transient increase in testicular weight was observed, and decreased
sperm counts and motility with histological abnormality in the testis
were found. These findings in VDR null mutant male mice were similar to
those in ER knockout mice (18, 21, 22). In the male gonads of ER
knockout mice, the fluid reabsorption in efferent ductules of the
testis was abnormal (22). In our study of VDR null mutant male mice,
estrogen deficiency appeared to cause gonadal insufficiencies by a
mechanism similar to that observed in ER knockout mice. In addition,
aromatase gene-deficient mice (ArKO) showed gonadal insufficiencies,
such as underdeveloped uteri and ovaries (23) and impaired
spermatogenesis (24). The phenotypes of gonads of ER and ArKO
paralleled those of the VDR null mutant male mice.
No histological abnormality was observed in the testes of male VDR null mutant mice supplemented with estrogen. The estrogen supplementation protected the testis of VDR null mutant mice from histological changes. These results strongly suggested that estrogen deficiency induced by VDR ablation is the cause of the abnormal spermatogenesis in VDR null mutant mice.
Decreases in the activity of P450arom and suppression of CYP19 gene
expression in both female and male gonads of the VDR null mutant mice
were demonstrated. The CYP19 gene encodes P450arom, the key enzyme for
estrogen biosynthesis, which dominantly influences the estrogen level.
Furthermore, the expressions of ER and ERß genes were normal in
gonads in VDR null mutant mice. These results indicated that the
estrogen-deficient state in VDR null mutant mice caused by decreased
P450arom activity depended on suppressed CYP19 gene expression.
It was reported that normalization of the serum calcium level restored fertility in vitamin D-deficient female and male rats (25, 26) and also prevented some phenotypic abnormalities in the VDR null mutant mice (9). To clarify the influences of severe hypocalcemia, calcium supplementation was performed in the VDR null mutant mice. A high calcium diet increased the serum calcium level to near that in wild-type mice. The normalization of the serum calcium level increased aromatase activity in the ovary to 60% of that in the wild-type animals. Furthermore, the expression level of the CYP19 gene was increased to 10-fold that in VDR null mutant mice without calcium supplementation. The high levels of LH and FSH after normalization of serum calcium meant that the endocrinological state remained abnormal.
Despite the abnormal endocrinological state, some VDR null mutant mice with a normal serum calcium level were fertile. This may explain why other VDR-ablated mice (27) did not show infertility, although the details of the gonadal functions were not reported. The serum calcium levels of these mice were much higher than those of our VDR null mutant mice [1.00–1.09 mM (82% of wild-type mice) vs. 5.36 ± 0.25 mg/dl (65% of wild-type mice)]. In human cases of vitamin D-dependent rickets type II, no gonadal insufficiencies were detected (28, 29). Calcium had been administered to these patients from an early phase. Normalization of the serum calcium level might therefore restore the infertility. It is possible that hypogonadism was generated by nonspecific disruption of the aromatase gene enhancer region. However, it is difficult to consider this possibility. Li model VDR-ablated mice (27) and our VDR null mutant mice revealed similar phenotypes, such as growth retardation, impairment of bone formation, and alopecia, though the phenotypes of the Li model were much milder. In addition, normalization of mineral ion homeostasis prevented the phenotypes, except for alopecia (9). The higher level of serum calcium might cause the milder phenotypes and fertility of the Li model.
It was recently reported that the P450arom activity of human choriocarcinoma cell lines was stimulated by 1,25-(OH)2D3 and that the VDR response element was identified in the CYP19 gene (30). This would suggest that vitamin D regulates the CYP19 gene directly. Using VDR null mutant mice, not vitamin D-deficient mice, we demonstrated that vitamin D acted to regulate estrogen biosynthesis: this regulation could not be explained by the calcitropic activities alone. These results indicated that vitamin D plays a role in estrogen biosynthesis partially by maintaining extracellular calcium homeostasis. However, direct regulation of the expression of the aromatase gene was also considered.
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Acknowledgments |
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Footnotes |
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Received June 29, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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