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Abstract

Vitamin D is a versatile signalling molecule with a well-established role in the regulation of calcium homeostasis and bone health. The spectrum of vitamin D target organs has expanded and the reproductive role of vitamin D is highlighted by expression of the vitamin D receptor (VDR) and enzymes that metabolize vitamin D in testis, male reproductive tract and human spermatozoa. The expression levels of VDR and CYP24A1 in human spermatozoa serve as positive predictive markers of semen quality, and VDR mediates a nongenomic increase in intracellular calcium concentration that induces sperm motility. Interestingly, functional animal models show that vitamin D is important for estrogen signalling and sperm motility, while cross-sectional studies support the positive association between serum 25-hydroxyvitamin D level and sperm motility in both fertile and infertile men. Expression of VDR and enzymes that metabolize vitamin D in fetal testis indicates a yet unknown role during development, which may be extrapolated from invasive testicular germ cell tumours where 1α,25-dihydroxyvitamin D induces a mesodermal differentiation of the pluripotent testicular cancer cells. Taken together, vitamin D signalling has a positive effect on semen quality, increases estrogen responsiveness and differentiates germ cell tumours. Future studies are needed to determine when 1α,25-dihydroxyvitamin D acts in a paracrine manner and whether systemic changes, which are subject to pharmacological modulation, could influence male reproductive function.
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1
University Department
of Growth and
Reproduction,
Rigshospitalet, Section
5064, Blegdamsvej 9,
2100 Copenhagen,
Denmark.
blombergjensen@
gmail.com
Vitamin D and male reproduction
Martin Blomberg Jensen
Abstract | VitaminD is a versatile signalling molecule with a well-established role in the regulation of calcium
homeostasis and bone health. The spectrum of vitamin D target organs has expanded and the reproductive
role of vitaminD is highlighted by expression of the vitaminD receptor (VDR) and enzymes that metabolize
vitaminD in testis, male reproductive tract and human spermatozoa. The expression levels
of VDR and CYP24A1 in human spermatozoa serve as positive predictive markers of semen quality, and
VDR mediates a nongenomic increase in intracellular calcium concentration that induces sperm motility.
Interestingly, functional animal models show that vitaminD is important for estrogen signalling and sperm
motility, while cross-sectional studies support the positive association between serum 25-hydroxyvitaminD
level and sperm motility in both fertile and infertile men. Expression of VDR and enzymes that metabolize
vitamin D in fetal testis indicates a yet unknown role during development, which may be extrapolated from
invasive testicular germ cell tumours where 1α,25-dihydroxyvitaminD induces a mesodermal differentiation
of the pluripotent testicular cancer cells. Taken together, vitaminD signalling has a positive effect on semen
quality, increases estrogen responsiveness and differentiates germ cell tumours. Future studies are needed
to determine when 1α,25-dihydroxyvitaminD acts in a paracrine manner and whether systemic changes, which
are subject to pharmacological modulation, could influence male reproductive function.
Blomberg Jensen, M. Nat. Rev. Endocrinol. advance online publication XX Month 2014; doi:10.1038/nrendo.2013.262
Introduction
The main functions of the testis are synthesis of sex hor-
mones and production of spermatozoa. Steroidogenesis
is performed by Leydig cells of the interstitium, whereas
spermatogenesis occurs inside the seminiferous tubules
(Figure1).1 Testicular functions require a multitude of
tightly regulated morphogenic and molecular events,
and the search for regulatory factors has been intense.
Most attention has been paid to effects occurring during
fetal life, as testicular development to a large degree pre-
determines adult testicular function. However, the adult
testis is also sensitive to various exposures and numer-
ous investigations have addressed the effect of nutrition,
hormones, pharmacological agents, endocrine disrupters
and vitamins on reproductive function.2
VitaminD is a versatile signalling molecule, and the
male reproductive organs are part of the expanding
palette of vitaminD targets, in addition to the classic
effects on bone, calcium and phosphate homeostasis.3,4
In the past decade, an ongoing debate concerning global
vitaminD deficiency in the general population5,6 has
prompted intensive research of the nonclassic effects of
vitaminD. In this Review, the spatial and temporal local-
ization of the vitaminD receptor (VDR) and enzymes
that metabolize vitaminD in the male reproductive
organs are discussed with the aim of conceptualizing
the role of systemic and local metabolism of vitaminD
in male fertility, sperm function, sex and reproduc-
tive hormone synthesis and testicular germ cell cancer.
Improved knowledge of the effects of vitaminD on male
reproduction will provide insights concerning the influ-
ence of classic bone factors on gonadal function and aid
understanding of human reproduction in general.
Vitamin D metabolism
The inactive form of vitamin D (cholecalciferol)
is synthesized in the skin following conversion of
7- dehydrocholesterol by ultravioletB radiation
(Figure2).7 Cholecalciferol must undergo two hydroxy-
lation events to form the active form of vitaminD, 1α,25-
dihydroxyvitaminD3. The first step is 25- hydroxylation
by the hepatic enzyme CYP2R1 followed by renal
1α-hydroxylation by CYP27B1, whereas CYP24A1 inac-
tivates all circulating forms of vitaminD.7 Subsequently,
1α,25-dihydroxyvitaminD3 binds and activates VDR that
forms a heterodimer with retinoidX receptor (RXR).
This complex recognizes a vitaminD response element
(VDRE) in the promoter region of target genes and regu-
lates transcription.8,9 The genomic ligand-binding pocket
of VDR mediates the transcriptional effects, but VDR
also has an alternative ligand-binding pocket that medi-
ates rapid nongenomic effects.8,10
VitaminD status is assessed by measuring the serum
concentration of 25-hydroxyvitaminD rather than
serum 1α,25-dihydroxyvitaminD3 because levels of
serum 25-hydroxyvitaminD are associated with rickets,
Competing interests
The author declares that he holds two patent applications
related to vitaminD and reproduction.
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hypocalcaemia, hypophosphataemia and serum levels of
parathyroid hormone (PTH).3,5,6 Hypocalcaemia often
accompanies vitaminD deficiency and is a major obstacle
to proper interpretation of proposed vitamin-D-mediated
effects. The effect of vitaminD on calcium and phosphate
homeostasis necessitates a tight regulation of the activity
of the enzymes that metabolize vitaminD; however, the
spatial expression of these enzymes is not restricted to the
liver and kidney.9 Extra-renal vitaminD metabolism is
not involved in calcium homeostasis, but rather in para-
crine and autocrine functions (for instance, the regulation
of cell cycle control) and, therefore, has a different system
of regulation than systemic vitaminD metabolism.9
Vitamin D and male reproductive organs
A prerequisite for being a vitaminD target organ is the
expression of VDR. However, the cellular responsive-
ness in target cells depends on not only circulating levels
of 1α,25-dihydroxyvitaminD3, but also cellular expres-
sion of CYP2R1, CYP27B1 and CYP24A1. The encoded
proteins activate or inactivate circulating cholecalciferol
or 25-hydroxyvitaminD, which modulates the intra-
cellular concentration of 1α,25-dihydroxyvitaminD3.
VDR and enzymes that metabolize vitaminD are con-
comitantly expressed in Sertoli cells, germ cells, Leydig
cells, spermatozoa and in the epithelial cells lining the
male reproductive tract (Figure1).4,11–17 The presence
of vitamin D metabolizing enzymes indicates that the
reproductive organs can modulate the local vitaminD
response, which is supported by the high concentration
of 25-hydroxyvitaminD, 1α,25-dihydroxyvitaminD3
and 24,25-dihydroxyvitaminD3 in rat testis and epi-
didymis compared with other organs following injection
of tritiated vitaminD progenitors.18 VDR has no testis-
specific splice variants19 and the high binding affinity
(VDRkd 50–100 pM)20–22 for 1α,25-dihydroxyvitaminD3
in the testis indicates that the concentration of 1α,25-
dihydroxyvitaminD3 in serum is adequate for gonadal
VDR activation.
Minor differences in the expression of VDR and
enzymes that metabolize vitaminD between species
Key points
VitaminD is metabolized in the male reproductive organs and the expression
levels of vitaminD receptor (VDR) and CYP24A1 in human spermatozoa are
positive markers for semen quality
1α,25-dihydroxyvitaminD3 induces a VDR-mediated increase in intracellular
calcium concentration in human spermatozoa invitro, leading to increased
motility and induction of the acrosome reaction in capacitated spermatozoa
Vdr-null mice and rodents with vitaminD deficiency develop impaired fertility
due to decreased sperm production and low sperm motility, which can only
partly be restored by calcium supplementation
Results from human association studies are in line with those in animal
models, as men with vitaminD sufficiency have a higher percentage of motile
spermatozoa than men with vitaminD deficiency
1α,25-dihydroxyvitaminD3 induces differentiation of embryonal carcinoma cells
invitro and invivo and increases cellular susceptibility to cisplatin invitro
The most important regulators of testicular vitaminD metabolism seem to
be fibroblast growth factor 23 and Klotho, other regulators are testosterone,
estradiol, calcium, phosphate and 1α,25-dihydroxyvitamin D3
exist: in humans, VDR is expressed mainly in germ
cells as well as fetal or immature Sertoli cells,4,23 whereas
in mice VDR is expressed in germ cells in addition
to both immature and mature adult Sertoli cells,24,25
which impedes translation of results from mice models
to humans. Interestingly, specific expression profiles
of VDR and enzymes that metabolize vitaminD in
human spermatozoa4,16,17,21 from healthy normospermic
and subfertile men distinguish the two populations of
sperm with high specificity and might thus prove clini-
cally relevant.21,26 In particular, the distinct localization
of CYP24A1 at the sperm annulus serves as a predic-
tive marker of good quality sperm,4,26 and the fraction
of CYP24A1-positive spermatozoa has been positively
associated with sperm count, concentration, motility and
morphology (Figure1).26
Importantly, >80% of CYP24A1-positive spermato-
zoa concomitantly express VDR in the neck and head
region.26 Co-expression of both proteins might be linked
to transcriptional induction during spermatogenesis,
wherein the VDR complex binds to one of the two positive
VDREs in the promoter region of CYP24A1.8 During the
later stages of spermatogenesis, cellular organelles are lost
and most histones are replaced by protamines that prevent
transcription and spermatozoa are thus almost tran-
scriptionally silent.27 Human spermatozoa are therefore
a unique model to study nongenomic effects exerted by
VDR (discussed later).10 The low expression levels of VDR
and CYP24A1 in spermatozoa from men with infertility
serves not only as a marker but implies unresponsiveness
to treatment or exposure to 1α,25-dihydroxyvitaminD3
in the male or female reproductive tract.26 Indeed, the
proposed functional consequences of low expression of
CYP24A1 in spermatozoa were supported by showing
an increase in sperm motility after treatment with 1α,25-
dihydroxyvitaminD3 invitro exclusively in healthy nor-
mospermic men and not in spermatozoa from men with
infertility3 who had a very low average proportion (<3%)
of CYP24A1-expressing sperm.26
The enzymes 1α-hydroxylase (CYP27B1) and
25-hydroxylase (CYP2R1) in particular, are highly
expressed in the testis compared with other tissues.28,29
CYP2R1 is expressed in both Leydig and germ cells4,23,25
and reduced CYP2R1 expression has been reported in
testis from men with impaired spermatogenesis, prob-
ably due to a decreased number of germ cells.30 These
patients also display a lower serum level of 25-hydroxy-
vitaminD than normospermic men, and it has been pro-
posed that testicular CYP2R1 is important for systemic
25- hydroxylation.30 This suggestion is attractive because
the relative expression of CYP2R1 is higher in human
testis than in the liver.28 Nevertheless, several studies
have shown that systemic 25-hydroxylation mainly takes
place in the liver. This theory is strongly supported by
the low circulating serum level of 25-hydroxyvitaminD
in mice following hepatectomy.31 However, an increase
in hepatic 25-hydroxylase has been observed following
castration in rats, which indicates that the testis might
contribute to, or at least produce, a regulator of hepatic
25-hydroxylase and therefore also influence serum levels
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of 25-hydroxyvitaminD.32
The transmembrane proteins LRP-2 (also known
as megalin) and cubulin facilitate cellular entry of the
protein-bound fraction of steroid hormones, includ-
ing 25-hydroxyvitaminD, in the kidney, prostate and
breast. Both proteins are expressed in the male repro-
ductive tract33 and might facilitate cellular entry of the
protein-bound forms of vitaminD (Figure3). VDR and
enzymes that metabolize vitaminD are also expressed in
the different segments of the epididymis (caput, corpus
and cauda), prostate and seminal vesicle of mice,20
rats,13 roosters14 and humans.4 The epididymis regulates
the composition of fluid surrounding the spermatozoa
during transition through the caput, corpus and cauda.
The concentration of calcium decreases, whilst the
concentration of phosphate increases in the fluid34 from
the proximal to the distal part of the epididymis, which
might be important for sperm maturation and involved
in the induction of sperm motility. During ejaculation,
the spermatozoa meet the secretions from the prostate
and seminal vesicle with a calcium concentration more
than twofold higher than serum,35 which might prepare
the spermatozoa for the environmental shift in the
female reproductive tract.
The effect of vitaminD in the efferent ducts and epi-
didymis might be comparable with that in the kidney
because they share developmental origin.36 The main
function of vitaminD in the kidney is transcellular
calcium transportation, which involves concerted action
of TRPV5, TRPV6, calbindin, PMCA1 and NCX1.3 The
hypothesized conserved function of vitaminD in both
organs is supported by the fact that TRPV6 is expressed
in the epididymis, and ablation of TRPV6 impairs epi-
didymal calcium absorption, which results in low sperm
motility and male infertility in mice.37
Vitamin D and gonadal hormones
Testosterone
Testosterone is produced by Leydig cells and is respon-
sible for primary and secondary male sex characteristics.
The concentration of testosterone is 100-fold higher in
the testis than in serum, and testosterone biosynthesis is
controlled by placental human chorionic gonadotropin
(hCG) in early fetal life until the pituitary starts to secrete
luteinizing hormone (LH).38 LH induces steroidogene-
sis by increasing cyclic AMP production and the intra-
cellular concentration of calcium ions (Ca2+) in Leydig
cells,38,39 and 1α,25-dihydroxyvitaminD3 might exert an
influence by modulating this calcium-dependent LH
response.
The testosterone:LH ratio is a good indicator of LH
sensitivity and Leydig cell function. In Vdr-null mice, a
tendency towards a low testosterone:LH ratio and Leydig
cell hyperplasia (potentially as a consequence of the low
ratio) were noted.25 However, serum levels of testoster-
one and LH were not significantly different between
wild-type and Vdr -null mice generated by two different
a
c d
e f
g h
b
Sp.z
Sp.t
Sp.g
Leydig
30
μm
30
μm
30
μm
Sp.c
NT
NT
CIS
NT
NT
CIS
30
μm
30
μm
S
Annulus
Figure 1 | Immunohistochemistry from the male
reproductive organs. a | Haematoxylin and eosin staining
of intratubular normal spermatogenesis with
spermatogonia (Sp.g.), Sertoli cells (S), spermatocytes
(Sp.c.), spermatids (Sp.t), spermatozoa (Sp.z) and
interstitial Leydig cells marked. b | Immunohistochemical
staining of CYP24A1 in human spermatozoa with a distinct
expression at the annulus separating the midpiece from
the tail. c | Alkaline phosphatase placental-like (PLAP) is a
marker of carcinoma insitu (CIS) cells, whereas there is no
detectable expression of PLAP in normal testis (NT).
d | Vitamin D receptor (VDR) expression in CIS and NT.
eh | Serial sections of OCT4, CYP27B1, VDR and
osteocalcin, respectively, in a nonseminona show that VDR
and enzymes that metabolize vitaminD in addition to a
VDR-regulated gene BGLAP (osteocalcin) are expressed in
cancer cells with none or low OCT4 expression.
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laboratories.
Correspondingly, hCG tests in three children with
hereditary 1α,25-dihydroxyvitaminD3-resistant rickets
(no functional VDR) elicited a normal testosterone
response,40 and infusion of 1α,25-dihydroxyvitaminD3 for
3days in healthy men caused no changes in serum levels of
testosterone or LH.41 Therefore, it is reasonable to assume
that no causal relationship exists between VDR activation
and serum levels of testosterone or LH sensi tivity. This
suggestion is in accordance with the comparable serum
concentrations of testosterone and LH in adolescents
and healthy young men with and without vitaminD
deficiency.42–44 However, in older men (>60years of age),
several cross-sectional studies45–48 have found positive
associations between serum levels of 25-hydroxy vitaminD
and testosterone and a seasonal variation in serum testos-
terone levels that matched the seasonal changes in serum
levels of 25-hydroxyvitaminD.46,49
The striking difference between young and older men
indicates an indirect effect of vitaminD, which was
supported by a cross-sectional study reporting signifi-
cant associations between serum levels of 25-hydroxy-
vitaminD and testosterone, which disappeared when
adjusting for health status and comorbidities.47 Further-
more, serum levels of 25-hydroxyvitaminD and sex
hormones decline with age, whereas serum levels of
sex hormone-binding globulin (SHBG; also known as
testis-specific androgen binding protein) increase.50 This
pattern might explain some of the discrepancy because
serum levels of 25-hydroxyvitaminD are positively asso-
ciated with those of SHBG in young men42,43 but not in
older men.46–48 Several factors regulate SHBG biosyn-
thesis, and a small randomized clinical trial tested the
combined effect of weight loss and chole calciferol sup-
plementation on levels of SHBG and testosterone in 54
obese men of an average age of 48years.45 The research-
ers found an increased serum level of SHBG, but no sig-
nificant changes in serum concentrations of testosterone
between placebo-treated and ch olecalciferol-treated
men.45 The presumed effect of vitaminD on SHBG in
the liver might be direct or indirect, but testicular Shbg
levels are comparable between Vdr -null mice and wild-
type littermates,25 which excludes a gonad-specific effect
of vitaminD on SHBG in mice.
VitaminD, in conjunction with PTH, regulates calcium
absorption in the intestine and excretion in the kidney.
Thus, serum levels of calcium can change as a result of
systemic changes in serum concentrations of vitaminD
and might, therefore, exert an indirect effect in the target
tissue. VitaminD deficiency is often accompanied by
hypocalcaemia, which might exert an influence on the
target tissue rather than having a direct VDR-mediated
effect. One example is the low serum level of testos terone
in vitamin-D- deficient rats, which increases 2–5-fold fol-
lowing injections of 1α,25-dihydroxyvitaminD3.51 By con-
trast, another study showed comparable serum levels of
testosterone between normocalcaemic vitamin-D-deficient
chickens and vitamin-D-replete chickens.52 The pro-
posed effect of calcium on steroidogenesis is biologically
grounded, as a low extracellular calcium level diminishes
the hCG- mediated effect on steroidogenesis and, there-
fore, constitutes a potential confounder.38 Another testos-
terone-inducing effect of vitaminD could be mediated by
osteocalcin, which is produced and secreted by the skel-
eton and the gene that encodes osteocalcin is regulated by
vitaminD.8 Osteocalcin has been proposed to stimulate tes-
tosterone production through activation of the promiscu-
ous receptor GPRC6A in mouse Leydig cells.53,54 However,
human data are limited. Osteocalcin might be positively
associated with serum concentration of testosterone but
is not a strong determinant of serum levels of testoster-
one in men.42,55,56 Combined, the effects of vitaminD on
testos terone production appear to be indirectly mediated
through calcium and phosphate homeostasis, SHBG or
osteocalcin production. The large randomized clinical
trials evaluating vitaminD supplementation to prevent
fractures might provide additional information in older
men while we await randomized clinical trials with serum
testosterone levels as a primary end point.
Cholecalciferol D3
Kidney
25-OHD3
1,25-(OH)2D3
GenomicNongenomic
Skin
Liver
7-dehydrocholesterol
CYP2R1
CYP27B1
CYP24A1
1,24,25-(OH)3D3
Target
tissue
VDR
Figure 2 | Systemic vitaminD metabolism. VitaminD synthesis normally starts in the
skin, where ultravioletB radiation initiates conversion of 7-dehydrocholesterol to
cholecalciferol. Cholecalciferol D3 is biologically inactive and undergoes two
hydroxylation steps before the active 1α,25-dihydroxyvitaminD3 is formed. Normally,
25-hydroxylation is mediated by the hepatic CYP2R1, whilst 1α-hydroxylation is
conducted by the renal CYP27B1. The active form of vitaminD, 1α,25-
dihydroxyvitaminD3, binds to the VDR and mediates rapid effects through the
alternative ligand binding pocket or genomic effects through the genomic pocket. All
the circulating forms of vitaminD are inactivated by 24-hydroxylation. Abbreviations:
25-OHD3, 25-hydroxyvitaminD3; 1,25-(OH)2D3, 1α,25-dihydroxyvitaminD3; 1,24,25-
(OH)3D3, 1,24,25-trihydroxyvitaminD3; VDR, vitamin D receptor.
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Estradiol
The CYP19A1 gene encodes aromatase, which converts
testosterone to estradiol, has multiple promoters and is
regulated differently depending on promoter activity in
the target tissue.57–59 1α,25-dihydroxyvitaminD3 binds to
VDREs in the promoter of CYP19A1 and exerts tissue-
specific regulation of aromatase by repressing or induc-
ing transcription in breast and bone, respectively.24,57,60
Several studies have investigated testicular aromatase
activity and identified promoterII as the main promoter
in the testis,61,62 although promoterI.4 might contribute
in the germ cells.63 Estrogens are not exclusively pro-
duced by Leydig cells as >60% of testicular aromatase
originates from germ cells.58 Estradiol concentration in
rete testis is therefore high,59 whereas the serum level of
estradiol is typically low in men.
Aromatase expression was induced by 1α,25-
dihydroxyvitaminD3 in immature rat Sertoli cells
invitro.24 Moreover, two Vdr -null mouse models (two
different strains of mice expressing a nonfunctional VDR
isoform in all tissues due to removal of exon2 of the Vdr
gene) convincingly showed a marked influence of 1α,25-
dihydroxyvitaminD3 on estrogen signalling in testis and
epididymis (Table1).25,60 However, the decreased tes-
ticular aromatase level and compensating increases in
serum levels of gonadotropins60 in the initial Vdr-null
mouse (produced in Tokyo, Japan) were not supported
in another Vdr -null mouse strain (produced in Leuven,
Belgium).25 Instead, serum levels of gonadotropins were
normal but the expression level of estrogen receptor (ER)
α and ERβ were altered. ERβ seemed to be a major target,
as heterozygous as well as homozygous mutant mice
with concomitant hypocalcaemia had low ERβ expres-
sion.25 The aberrant estrogen response was supported
by showing an epididymal upregulation of the estrogen
regulated gene aquaporin9.
Hypocalcaemia complicates the interpretation of the
molecular phenotype of Vdr -null mice, as it has been
demonstrated that low serum levels of calcium induce
testicular aromatase transcription and activity in rats.64
This observation contrasts with the reported decline in
aromatase transcription and activity in the first study
of Vdr-null mice.60 Moreover, the reproductive pheno-
type of the Vdr-null mouse model60 does not resemble
the inconsistent and late-onset impaired male fertility
observed in the three different Cyp19a1-null mice.58
Instead, Vdr-null mice have some similarities with Erα-
null mice, which are characterized by low sperm motility
and infertility due to decreased fluid reabsorption in rete
testis and caput epididymis.65,66 However, the complex
molecular phenotype with low Cyp19a1 and Erβ expres-
sion and high epididymal ERα in Vd r-null mice indicates
that local estrogen signalling is altered. A relationship
between VDR and estrogen in the male reproductive
organs is further supported by expression of the estrogen
receptors and VDR in the same cells,36 and by the revers-
ible decrease in spermatogenesis and sperm motility in
Vdr -null mice60 following supplementation with calcium
and estrogen.
The systemic effects of aberrant estrogen signalling
due to loss of VDR are less pronounced than those at
the gonadal sites because serum levels of estradiol
were not significantly different between Vdr -null mice
and their wild-type littermates in two different mouse
strains.25,60 Accordingly, no significant associations
were found between serum levels of estradiol and
25-hydroxy vitaminD in three cross-sectional studies of
healthy young men.42–44 However, in older men47 a nega-
tive association between serum levels of estradiol and
25-hydroxyvitaminD was reported, which indicates that
extra-gonadal conversion of testosterone by aromatase,
which mainly occurs in adipose tissue, might contribute
to serum levels of estradiol.
Testicular peptide hormones
InhibinB production reflects the close interaction
between Sertoli cells and spermatocytes in the adult
testis and is therefore used clinically as an indica-
tor of spermatogenesis.67 By contrast, anti-Müllerian
Cholecalciferol D3
25-OHD31,25-(OH)2D3
CYP27B1
VDR
DBP
Megalin
Cubulin
VDR RXR
Transcription
PLC
PKA
Calcium
Chloride
CYP24A1
Testosterone
Estradiol
VDRE
Nucleus
1,24,25-(OH)3D3
PTHrP
Estradiol
Low PO4
2–
1,25(OH)2D3
FGF23
High Ca2+
PTHrP
Low PO4
2–
1,25(OH)2D3
FGF23
High Ca2+
VDR
Testosterone
CYP2R1
Figure 3 | Factors influencing cellular vitaminD metabolism in the male
reproductive organs. The circulating forms of vitaminD (cholecalciferol,
25-hydroxyvitaminD and 1α,25-dihydroxyvitaminD3) diffuse freely across the
plasma membrane, whereas the protein-bound fraction (bound to DBP) are
transported actively into the cell by megalin or cubulin. The cell expresses the
enzymes that metabolize vitaminD (CYP2R1, CYP27B1 and CYP24A1) and are
thus capable of activating or inactivating all circulating forms of vitaminD.
1α,25-dihydroxyvitaminD3 elicits the effects through the VDR. VDR mediates rapid
actions when situated at the membrane or in the cytoplasm through ion channels
or modulation of second messengers. VDR heterodimerizes with RXR and binds to
a VDRE in the promoter region of the target genes and regulates transcription. The
regulators of local vitaminD metabolism in the male reproductive organs and their
proposed targets and actions are marked on the figure (stimulators , inhibitors ).
Abbreviations: 25-OHD3, 25-hydroxyvitamin D3; 1,25-(OH)2D3, 1α,25-
dihydroxyvitamin D3; 1,24,25-(OH)3D3, 1,24,25-trihydroxyvitaminD3; DBP, vitamin D
binding protein; FGF23, fibroblast growth factor 23; PKC, protein kinase C; PLC,
phospholipase C; PTHrP, parathyroid hormone-related protein; RXR, retinoid X
receptor; VDR, vitamin D receptor; VDRE, vitamin D response element.
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hormone (AMH) is produced predominantly by imma-
ture Sertoli cells and is involved in the development of
the male reproductive tract, whereas its role in adult
life is not resolved.1 A cohort study published in 2012
reported a positive association between serum levels of
25-hydroxyvitaminD and AMH, and showed that AMH
production was modulated by cholecalciferol supple-
mentation in adult men, but not in boys.68
Direct stimulation of AMH production by 1α,25-
dihydroxyvitaminD3 is plausible because the AMH
promoter contains a VDRE, and AMH is solely pro-
duced by immature human Sertoli cells that express
VDR, unlike mature human Sertoli cells. VDR is also
expressed in prostate cancer cells, in which 1α,25-
dihydroxyvitaminD3 has been shown to induce AMH
transcription invitro.69 Of note, the proposed influence
of vitaminD on testicular AMH production in humans
has not been corroborated by functional animal studies.
No statistically significant changes in Amh expression
were observed between Vdr-null, heterozygous and
homozygous wild-type littermates.25 AMH is exclusively
expressed in immature Sertoli cells, whereas mature
Sertoli cells produce and secrete inhibinB and SHBG.
InhibinB exerts negative feedback on FSH secretion,
whereas SHBG determines free hormone concentration
in the male reproductive tract. No differences were found
in the transcriptional levels of testicular Shbg or InhibinB
levels and, in accordance, comparable serum FSH and
LH levels were found between Vdr -null and wild-type
control mice.19,25 Moreover, in human cohort studies
in healthy men, no associations were found between
serum concentrations of 25-hydroxyvitaminD and FSH
or inhibinB,22,42–44 hence VDR seems to be dispensable
for testicular SHBG or Inhibin B expression,24 whilst the
weak association with AMH in humans requires valida-
tion. Insulin-like factor3 (INSL3) is a peptide hormone
produced by Leydig cells and is important for inducing
testicular descent during development.70 Insl3 was not
differentially expressed between Vdr -null mice and their
wild-type littermates and is therefore not dependent on
VDR expression.25 Progesterone synthesis is induced by
1α,25-dihydroxyvitaminD3 in the placenta and ovaries,71
but the putative influence of vitaminD on progesterone
production in the Leydig cells remains to be investigated
in both humans and animals.
Vitamin D and male fertility
An estimated 10–15% of couples are infertile, and the
causative contribution seems to be equal between the
sexes.72 Male fertility is evaluated routinely by semen
analysis and to date no evidence-based treatment exists
for impaired semen quality, which is predominantly idio-
pathic. A beneficial effect of vitaminD has been shown
in rats inseminated with sperm from male rats with
vitaminD deficiency, which produced 71% fewer preg-
nancies compared with sperm from vitamin-D- sufficient
rats.73 A follow-up study suggested that fertility was
restored by correcting the concomitant hypo calcaemia
in the vitamin-D-deficient rats and therefore not a
direct effect of vitaminD.74 However, re-assessment of
the data revealed that pregnancy rates remained 43%
lower after insemination with sperm from normo-
calcaemic vitamin-D-deficient rats than in normo-
calcaemic vitamin-D-replete rats.74,75 Thus, a direct effect
of vitaminD on male fertility is plausible.
The low fertility rates in vitamin-D-deficient male
Table 1 | VDR-regulated proteins and pathways in male reproduction
Pathway Proteins and ions Tissue Reproductive effect
Calcium and phosphate
homeostasis
TRPV5,118 TRPV6,37 calbindin
1–3,52,118 PMCA1–4,119 NCX1–3,120
NPT2a–c,121 CaSR122
Testis and/
or epididymis
Putative involvement in transcellular calcium
and phosphate transportation and calcium
storage in germ cells, Leydig cells, efferent
ducts and epididymis
Reproductive hormones CYP19A1,24,25,60 ERα,25 ERβ,25
AMH,25,68,69 IGF-BP38,125
Testis and/
or epididymis
Regulation of estrogen signalling in testis,
efferent ducts and epididymis
Potential regulation of testicular AMH and
IGF-BP3 synthesis
Cell cycle control p21,93 p27,93 p53,93 p73,93 FOXO193 Testis Regulation of TGCT proliferation, differentiation
and cisplatin resistance
Pluripotency and germ
cell layer commitment
OCT4,23 NANOG,23 SNAI123 Testis Regulation of pluripotency factors and
mesodermal differentiation in TGCT
Possible role in fetal development
Regulators of testicular
and epididymal vitaminD
metabolism
PTHrP,106 FGF23,23,123 Klotho,124
CYP27B1,25 CYP24A125,26
Testis Suggested paracrine genetic regulators of
testicular vitaminD metabolism
Osteogenic signalling RUNX2,23,131 SSP1,23 BGLAP23 Testis Osteogenic differentiation or transdifferentiation
in dysgenetic testis with germ cell tumours
Mixed ABCA1,126 GGT127 Testis Cholesterol homeostasis and Sertoli cell
function
Nongenomic Ca2+, Cl, K +-ux,21,22,128 PKA, PKC,
PLC22,25,26 MAPK118
Testis Rapid nongenomic effects mediated by VDR
The genes of the proteins included in the table are all directly or indirectly regulated by VDR but only some of them have a validated vitaminD response element
in the promoter region. Abbreviations: AMH, anti-Müllerian hormone; FGF23, fibroblast growth factor 23; TGCT, testicular germ cell tumour; VDR, vitaminD
receptor.
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animals (such as mice, rats and wild boars) seems to be
caused by impaired sperm motility and, occasionally,
poor sperm morphology.60,73,76–78 Accordingly, a compre-
hensive reproductive investigation in a Vdr-null mouse
model revealed a marked decrease in sperm number
(40%) and sperm motility (ninefold), which resulted in
infertility.60 Semen quality and fertility improved after
calcium supplementation but was only normalized after
supplementation with both calcium and estrogen.60 This
finding indicates that male fertility, besides being influ-
enced by vitaminD, is susceptible to systemic changes in
calcium and estradiol. Interestingly, testicular histology
was grossly normal in two out of three Vdr -null mouse
strains19,25,60 and in chickens with vitaminD deficiency.52
However, impaired spermatogenesis was detectable in
seminiferous tubules concomitantly with low aroma-
tase expression and activity in another Vdr-null mouse
strain.60 The low ERβ and increased ERα expression in
the reproductive organs of another Vd r -null mouse model
did to some extend corroborate the diminished estrogen
responsiveness, which ultimately might be mediating the
influence of vitaminD on sperm motility (Table2).25,37,59
Knowledge of the reproductive effects of vitaminD
in adulthood are largely based on data from cross-
sectional studies, in which men with vitaminD defi-
ciency (<25 nmol/l) or insufficiency (<50 nmol/l)
had significantly lower sperm motility than men with
vitaminD sufficiency.26,22,79 The positive association with
motility has been shown in young men from the general
population,22 fertile men79 and men with infertility.26,79
Another cohort study comprising mainly vitamin-D-
sufficient young men43 reported an unadjusted nonsig-
nificant (P = 0.06) positive trend between serum levels of
25-hydroxyvitaminD and sperm motility. Furthermore,
a small study showed that men with serum levels of
25-hydroxyvitaminD of 50–125 nmol/l had more motile
sperm than men with levels <50 nmol/l or >125 nmol/l.44
Sperm morphology was positively associated with serum
levels of 25-hydroxyvitaminD in two studies,22,79 whereas
all larger studies22,26,43,79 found no association with total
sperm count, sperm concentration or serum levels of
inhibinB or FSH (Table2).
The effect of 1α,25-dihydroxyvitaminD3 on sperm
motility might be mediated through the aforementioned
epididymal changes, but 1α,25-dihydroxyvitaminD3
is also a potent inducer of nongenomic effects in
human spermatozoa.8 VDR elicits a rapid increase
in intracellular Ca2+ concentration through inositol
trisphosphate(IP3)-mediated Ca2+-release from an
intracellular IP3-receptor-gated calcium store in the
neck of human spermatozoa (Figure4).10,21,26,80 The
nongenomic nature of this response was evident due
to the rapid kinetics (seconds) initiated in the com-
partment expressing VDR17 and validated by using the
6-cis-locked agonist 1α,25-dihydroxylumisterol for rapid
effects mediated by VDR. 1α,25-dihydroxylumisterol
and 1α,25-dihydroxyvitaminD3 induced a similar effect,
whereas pretreatment with the nongenomic competi-
tive VDR antagonist 1β,25-dihydroxyvitaminD3 inhib-
ited the effect of 1α,25-dihydroxyvitaminD3.22,81 The
increase in intracellular Ca2+ concentration leads to
induction of sperm motility in both capacitated21 and
uncapacitated sperm,22,26 improves sperm–egg binding
invitro82 and triggers the acrosome reaction,22 which is
a prerequisite to fertilize the oocyte. The invitro effects
of 1α,25-dihydroxyvitaminD3 corroborate the effect on
sperm motility shown in both functional animal studies
and human association studies. The suggested relation-
ships between vitaminD and fertility are also supported
by epidemiological data showing a seasonal variation
in the conception rate in the northern countries of the
Northern Hemisphere,83 which peaks in the summer
and matches the seasonal variation in serum levels of
25-hydroxyvitaminD.
VitaminD is not only important in adulthood, as
VDR is already expressed in human gonocytes, imma-
ture Sertoli and Leydig cells from gestational week16,23
which strongly indicates an early effect of vitaminD in
the developing gonad. However, the role of vitaminD
in fetal life is largely unexplored and can so far only be
extrapolated from studies on testicular germ cell cancer
cells that resemble embryonic stem cells.
Vitamin D and testicular cancer
Testicular germ cell tumours (TGCT) are the most fre-
quent type of solid tumour in young men, and their
embryonic stem cell-like totipotentiality manifested
by the ability to differentiate into all tissue types sepa-
rates them from somatic cancers.84 TGCTs originate
from a precursor, carcinoma insitu (CIS),85 which has
been identified as transformed fetal gonocytes or pri-
mordial germ cells (Figure1).86 After puberty, CIS cells
undergo malignant transformation and form either an
invasive seminoma, which retains germ cell characteris-
tics, or a nonseminoma that contains a de-differentiated
Table 2 | Reproductive effects of vitamin D
Reproductive
function
Species
Mouse Rat Human
Fertility
Conception 60,129 73–75 83
Time to pregnancy 60,129 73–75 ND
Littersize 25,60 73–75 NA
Semen quality
Concentration 60,126 73–75 22,26,43,44,79
Motility 60 73–75 22,26,43,44,79
Morphology ND ND 22,26,43,44,79
Reproductive hormones
Testosterone 19,25 51 40–48
Estradiol 25,60 24 42–44
AMH 25 ND 68,69
Inhibin B 25 ND 22,43
, increase; , no effect. One arrow indicates an association proposed by
at least one study or conflicting data with the majority of studies indicating
this effect. Two arrows indicate that at least two different studies show a
reproducible effect. Abbreviations: AMH, anti-Müllerian hormone; NA, not
available; ND, not determined; TTP, time to pregnancy.
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embryonic component (embryonal carcinoma) resem-
bling embryonic stem cells and more differentiated
somatic (teratomas) or extra-embryonic (yolk sac or
chorio carcinoma) components.87
Cellular expression of VDR and enzymes that metabo-
lize vitaminD changes during the malignant transforma-
tion of most somatic cancers,88,89 and the changes have
been suggested to be implicated in cancer growth and
progression as 1α,25-dihydroxyvitaminD3 promotes dif-
ferentiation and decreases proliferation of most cancer
cells.90 The VDR and enzymes that metabolize vitamin D
have a markedly high expression in CIS, but the expres-
sion level decreases when CIS progress to invasive semi-
nomas, and cellular expression of VDR and enzymes that
metabolize vitaminD are lost during the transition to
embryonic carcinoma (EC).23 In other words, the pres-
ence of vitaminD metabolism machinery in TGCTs
reflects the state of differentiation and decreases during
de-differentiation from CIS to EC.
The pluripotent EC is characterized by loss of VDR
and enzymes that metabolize vitaminD, but expres-
sion of VDR and enzymes that metabolize vitaminD
is reintroduced when the EC cells start to differenti-
ate into all types of tissue (teratomas) concomitantly
with the downregulation of pluripotency factors
(Table1).23 Correspondingly, treatment with 1α,25-
dihydroxyvitaminD3 downregulates pluripotency factors
in seminoma-derived TCam2 cells and EC-derived
NTera2 cells invitro and induces a mesodermal transi-
tion towards an osteogenic differentiation of the NTera2
cells invitro and in a NTera2 xenograft mouse model.23
This finding is intriguing because NTera2 cells are pluri-
potent cells derived from a pulmonary EC metastasis
that normally undergoes terminal differentiation into a
neuron-like cell following treatment with retinoic acid.
By contrast, TCam2 cells express pluripotency markers
such as OCT4 and NANOG but retain germ cell charac-
teristics and are unable to complete differen tiation with
retinoic acid.23 The pro-differentiating effect of 1α,25-
dihydroxyvitaminD3 was illustrated by downregulation
of OCT4 and NANOG and concomitant upregulation
of SNAI1, brachyuri, osteocalcin, osteopontin and fibro-
blast growth factor23 (FGF23) (Figure1).23 Surprisingly,
the changes in gene and protein expression had no effect
on tumour growth. Instead, the presence of classic
bone markers and alkaline phosphatase in TGCTs from
patients, xenograft tumours and cancer cells invitro indi-
cates that some testicular cancer cells possess the capacity
to differentiate and initiate the bone-specific mineraliza-
tion process.23 This finding might be clinically relevant
in patients with testicular microlithiasis, which can be
detected with ultrasonography due to depositions of
hydroxyapatite,91 and is a frequent finding in the tissue
adjacent to TGCTs,92 and might be linked with the osteo-
genic differentiation of some testicular cancer cells.
Most of the known anti-proliferative effects of 1α,25-
dihydroxyvitamin D3 invitro are mediated by cyclin-
dependent kinase inhibitors such as p21, p27, p63, p73
or by the repression of pro-survival proteins leading
to increased apoptosis.8,9,90 In NTera2 cells, 1α,25-
dihydroxyvitaminD3 induced a twofold to fivefold
increase in p21, p27, p53, p73 and FOXO1 transcrip-
tion, but the changes in gene transcription were not
reflected by the fraction of surviving or viable cells.93
1α,25-dihydroxyvitaminD3 or cholecalciferol treatment
have a growth inhibitory effect and increases the sus-
ceptibility to cisplatin in many somatic cancers.93 This
phenomenon might be relevant in TGCTs as the VDR-
regulated gene p21 largely determines cisplatin resistance
in testicular cancer.94 Indeed, co-treatment with cisplatin
and 1α,25-dihydroxyvitaminD3 reduced the propor-
tion of viable and surviving cells more than cisplatin
alone in both seminoma-derived and EC-derived cells
lines invitro. Interestingly, 1 M cisplatin plus 100 nM
1α,25-dihydroxyvitaminD3 caused a larger reduction in
surviving EC-derived cells than 5 M cisplatin treatment
alone. The proposed cisplatin-sparing effect of 1α,25-
dihydroxyvitaminD3 is of clinical interest because men
with TGCTs are young at the onset of disease and some
experience long-term adverse effects of cisplatin treat-
ment, such as early cardiovascular complications following
curative treatment of advanced TGCTs.95 Unf or tunately,
the antitumour effect of 1α,25-dihydroxyvitaminD3 plus
cisplatin was not significantly improved compared with
cisplatin alone in a xenograft mouse model,93 although
animals treated with 1α,25-dihydroxyvitaminD3 plus
cisplatin tended to have a larger reduction in tumour
burden than animals that received the monotherapy. The
study was compromised by the high dose of cisplatin
used (6 mg/kg per week) because it proved very efficient
and eradicated the large tumours rapidly.93 Therefore,
future investigations should reduce cisplatin dosage to
elucidate a potential cisplatin sparing effect of 1α,25-
dihydroxyvitaminD3 invivo.
Regulation of vitamin D metabolism
The systemic regulators of vitaminD metabolism
(PTH, FGF23, calcitonin, calcium, phosphate, 1α,25-
dihydroxyvitamin D3 and estradiol96,97) predominantly
target the renal 1α-hydroxylase (CYP27B1), but some
RNE
VDR
Head
Neck
Midpiece
Annulus
Tail
PLC
IP3R
Ca2+
[Ca2+]i
Ca2+
SOCE
Acrosome
Ins(1,4,5)P3
Figure 4 | Proposed mechanism for the nongenomic effect of VDR in human
spermatozoa. 1α,25-dihydroxyvitaminD3 activates VDR in the neck region that
elicits PLC activation and generation of IP3 production that subsequently opens
IP3R gated calcium channels in the RNE and increases intracellular Ca2+
concentration. The initial Ca2+ release from RNE might be supported by SOCE, but
this seems not to be through l-type channels as nifedipine is unable to influence
the 1α,25-dihydroxyvitaminD3-mediated increase in [Ca2+]i. Abbreviations: [Ca2+]i,
intracellular concentration of calcium ions; PLC, phospholipase C; RNE, redundant
nuclear envelope; SOCE, store-operated calcium entry; VDR, vitaminD receptor.
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of these factor have an additional effect on 24-hydroxy-
lase (CYP24A1) that amplifies their effect on vitaminD
metabolism. Extra-renal vitaminD metabolism is regu-
lated differently than systemic vitaminD metabolism
probably due to the lack of effect on systemic calcium and
phosphate homeostasis by extra-renal vitaminD metabo-
lism.90,97 Thus, systemic regulators might be less impor-
tant for extra-renal vitaminD metabolism, and paracrine
and autocrine factors such as IFN-γ, IGF-1, BMPs, TGF-β
and 1α,25-dihydroxyvitaminD3 might be more potent
regulators depending on the expression profile of the
specific regulators in the target tissue.96,97 The effects of
paracrine vitaminD signalling differs between organs
and extrapolating proposed regulators of extra-renal
vitaminD metabolism from one organ to another might
not be applicable, as illustrated by the various effects of
FGF23 on different tissues with vitaminD metabolism.98
The testis has a unique feature in that the male germ
cells express Klotho,99,100 which interacts with FGFR1
or FGFR3 to create a specific receptor for FGF23.101 In
the kidney, FGF23 is a key regulator of the sodium–
phosphate transporter NPT2A102 and inhibits 1α,25-
dihydroxyvitaminD3 production by lowering CYP27B1
and inducing CYP24A1 expression. FGFR1 and FGFR3
are also expressed in male germ cells,103 which could
render the testis a FGF23 target (Figure2). In fact, both
Klotho and Fgf23 knockout mice display male infertility,104
which highlights the importance of FGF23 signalling in
the testis. Moreover, Fgf23-null mice had the highest (10-
fold) increase in 1α-hydroxylase (CYP27B1) expression
in the testis compared with other nonrenal tissues such as
heart, aorta, spleen, bone and intestine.98 Thus, the bone-
secreted FGF23 acts upstream of CYP27B1 in the testis
and it is reasonable to imply that testicular CYP24A1 is
also a target of FGF23.98 Interestingly, FGF23 is highly
expressed in EC-derived cell lines, suggesting that TGCTs
in the testis results in an ectopic FGF23 production.23 In
turn, if this ectopic FGF23 is secreted it could influence
vitaminD metabolism and phosphate homeostasis in the
normal Klotho-expressing testicular tissue adjacent to the
tumour and might thus be involved in the formation of
testicular microlithiasis.
The counterpart to FGF23 is PTH or PTH-related
peptide (PTHrP),102 which normally increases the produc-
tion of 1α,25-dihydroxyvitaminD3. PTHrP is produced
locally by germ cells and the specific receptors P THR1 and
PTHR2 are also expressed in the testis.105,106 Nevertheless,
the effect of PTH and PTHrP on testicular vitaminD
metabolism remains to be elucidated. Calcitonin and
calcitonin-related peptide are important for male repro-
ductive function, but are also known stimulators of renal
vitaminD metabolism.107 However, both receptors are
only expressed in Leydig cells and spermatozoa, which
limits the effect on intratubular vitaminD metabolism.107
Sex steroid hormones are known regulators of
1α-hydroxylase, but they also induce sex-specific changes
in microsomal 25-hydroxylase activity in rats following
castration or hypophysectomy108,109 and in healthy men
following injection of anabolic steroids.110 The up to 100-
fold higher testicular concentrations of testosterone and
estradiol might thus induce an even stronger influence
on testicular and epididymal vitaminD metabolism com-
pared with the reported effects of serum sex hormone
levels on systemic vitaminD metabolism.1,111 The global
expression of ERs in the male reproductive organs36,112
indicates a regulatory role of estradiol in all compart-
ments of the reproductive tract, although a testis-specific
regulation of the metabolizing enzymes by ERs remains to
be demonstrated.58,113 By contrast, the androgen receptor
(AR) has a restricted expression pattern. AR is expressed
in the epithelial cells lining the reproductive tract and
peritubular cells, and the expression profile in Sertoli cells
is complex. Immature human Sertoli cells1 that express
VDR do not express AR, whereas adult Sertoli cells that
express AR have almost no expression of VDR.4,23 VDR
expression might thus decrease simulta neously with the
induction of AR expression during Sertoli cell matura-
tion.1 It is possible that AR regulates VDR expression
directly in the Sertoli cells because testosterone lowers
VDR expression in NTera2 cells,114 which strongly sup-
ports a regulatory function of testosterone on testicular
vitaminD metabolism (Figure2).
Functional studies have provided substantial evi-
dence for a direct 1α,25-dihydroxyvitaminD3-mediated
downregulation of CYP27B1 and upregulation of
CYP24A1.115,116 This finding indicates that local
vitaminD metabolism is stongly influenced by a tes-
ticular feedback system. The high CYP27B1 expression
and low CYP24A1 expression in the testis from Vdr-
null mice confirmed that 1α,25-dihydroxyvitaminD3
depends on a functional VDR to modulate the expres-
sion of the metabolizing enzymes.25 The intracellular
1α,25-dihydroxyvitaminD3 concentration seems to
be an important factor for testicular function and it is
therefore plausible that circulating levels of 25-hydroxy-
vitaminD or 1α,25-dihydroxyvitaminD3 in serum could
influence the expression level of testicular enzymes that
metabolize vitaminD and VDR. However, infusion
of 1α,25-dihydroxyvitaminD3 in rats did not change
VDR expression in the testis117 and serum levels of
25-hydroxyvitaminD were not associated with CYP24A1
expression in spermatozoa in a small cohort of healthy
and infertile men.26 Thus, serum levels of vitaminD
metabolites cannot be regarded as strong determinants
of testicular vitaminD metabolism.
Conclusions
VitaminD is metabolized in the testis and male
reproductive tract of both rodents and humans. A
functional VDR seems to be dispensable for normal
testicular develop ment in mice; however, mutant mice
with vitaminD deficiency and Vdr -null mice develop
reduced male fertility later in life. The decreased fertility
is mainly caused by impaired sperm motility and gamete
quality rather than diminished sperm production, as
illustrated by the grossly normal testicular histology in
two out of the three VDR-null strains and in CYP27B1-
knockout mice. The global expression of VDR implies
that the effect on semen quality can be mediated directly
in the germ cells, indirectly by modulating Leydig cell
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function or through changes in epididymal function,
as indicated by the aberrant estrogen signalling in Vdr -
null mice. Extrapolating results from mice to humans
is particularly problematic in the reproductive field
as human fertility potential is much lower than that
in rodents and even key signalling pathways such as
progesterone-mediated activation of CATSPER is not
conserved between species.
In humans, vitaminD is metabolized in the fetal testis
throughout development. The presence of vitaminD sig-
nalling in the fetal gonad is intriguing but the exact role
remains to be shown. Some clues can be extrapolated
from the pro-differentiation effects found in TGCTs, in
which 1α,25-dihydroxyvitaminD3 is a potent inducer
of mesodermal differentiation towards an osteogenic
phenotype of cancer cells with embryonic stem cell
characteristics. 1α,25-dihydroxyvitaminD3 might also
regulate cell cycle control in normal adult germ cells, but
after puberty vitaminD metabolism seems to be more
important for postmeiotic germ cell maturation, and the
VDR-dependent expression of CYP24A1 in human sper-
matozoa can be used clinically as a positive predictive
marker of semen quality.
The presence of VDR and enzymes that metabolize
vitaminD in human spermatozoa also has functional
consequences, convincingly shown by the rapid non-
genomic 1α,25-dihydroxyvitaminD3-mediated increase
in intracellular calcium concentration that induces
sperm motility. This finding corroborates a functional
relationship between vitaminD and sperm motility,
which already at this stage could be tested during invitro
fertilization. Interestingly, serum 25-hydroxyvitaminD
concentrations are positively associated with sperm
motility in both healthy men and those with infertil-
ity, and future studies will show whether sperm motil-
ity could be improved by supplementing vitaminD to
vitamin-D-deficient fertile or infertile men.
However, most of the vitamin-D-mediated effects
in humans are exclusively paracrine effects, wherein
VDR acts as a transcription factor. The reproductive
changes in gene transcription or protein expression
are rarely reflected systemically and are not influenced
by serum concentration of 25-hydroxyvitaminD. The
putative cisplatin-sparing effect of combining cisplatin
with 1α,25-dihydroxyvitaminD3 in the treatment of
advanced TGCTs was not verified in the invivo xeno-
graft model but should be evaluated in well-designed
invivo studies with a lower cisplatin dose than that pre-
viously used. Finally, emerging evidence corroborates an
endocrine interaction between the skeleton and gonads,
and suggests that 1α,25-dihydroxyvitaminD3, besides a
direct gonadal effect, is a potent regulator of many bone-
specific proteins, including osteocalcin and FGF23 that
might modulate testicular function indirectly.
1. Sharpe, R.M., McKinnell, C., Kivlin, C. & Fisher,J.S.
Review criteria
A search for original articles published until May 2013
focusing on vitaminD and male reproductive function
was performed in PubMed. The search terms used were
“reproduction”, “male reproduction”, “sperm”, “testis”,
“gonad”, “fertility”, “epididymis”, “seminal plasma”,
“sex hormones”, “testosterone”, “estradiol”, “AMH”,
“inhibinB” “INSL3”, “testis cancer”, “testicular germ
cell tumour” in combination with vitaminD. All papers
identified were English-language, full-text papers.
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cells, and their relevance to disorders of testis
function in adulthood. Reproduction 125, 769–
784 (2003).
2. Mortimer, D. et al. What should it take to
describe a substance or product as ‘sperm-
safe’. Hum. Reprod. Update 19 (Suppl. 1), i1–i45
(2013).
3. Bouillon, R. et al. Vitamin D and human health:
lessons from vitamin D receptor null mice.
Endocr. Rev. 29, 726–776 (2008).
4. Blomberg Jensen, M. et al. Vitamin D receptor
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Acknowledgements
I am very grateful to S. Dissing, N.E. Skakkebaek,
A. Juul, A. Jørgensen, J.E. Nielsen, T. Svingen
and especially E. Rajpert De-Meyts for constructive
comments.
REVIEWS
... Also, it improves sperm-egg binding in vitro and triggers the acrosome reaction, which is a prerequisite to oocyte fertilization. 29,30 Studies with VDR-null mice and rodents demonstrated that those with vitamin D deficiency had impaired fertility due to compromised sperm motility and, occasionally, poor sperm morphology. Also, it has been demonstrated that the lower fertility rates can only partly be restored by calcium supplementation. ...
... Also, it has been demonstrated that the lower fertility rates can only partly be restored by calcium supplementation. 30 A presumed link between vitamin D serum levels and semen quality has been evaluated in several human studies. A positive correlation between serum levels of vitamin D and sperm motility was found in studies both in young healthy and in infertile men. ...
... Monteiro AM / Rev Port Endocrinol Diabetes Metab. 2018;13(1):[26][27][28][29][30] ...
... Crosstalk between endocrine factors, including FGF23 and 1,25(OH) 2 D 3 , and male reproductive function has, in recent years, been a subject of increasing scientific focus (Blomberg Jensen, 2014;Bøllehuus Hansen et al., 2020;Yahyavi et al., 2024). ...
Article
STUDY QUESTION Is serum phosphate linked with semen quality and reproductive hormones in infertile men? SUMMARY ANSWER Hypophosphatemia is a frequent finding in infertile men and is associated with lower number of motile sperm. WHAT IS KNOWN ALREADY Phosphate is available in fluid from all segments of the male reproductive tract in concentrations manyfold higher than in serum. However, the role of phosphate in male fertility is largely unknown. STUDY DESIGN, SIZE, DURATION This cross-sectional study included 1242 men referred due to infertility between January 2017 and May 2020 at the Department of Growth and Reproduction, Rigshospitalet, Copenhagen. PARTICIPANTS/MATERIALS, SETTING, METHODS Each man underwent a physical examination, had semen parameters assessed, and had blood analyzed prospectively for concentrations of phosphate, ionized calcium, alkaline phosphatase, parathyroid hormone, serum 25-hydroxyvitamin D (25OHD), and reproductive hormones. After 246 men were excluded due to serious comorbidities, 1242 were included in the analyses. MAIN RESULTS AND THE ROLE OF CHANCE Infertile men have a high prevalence of mild (25.5%, 0.66–0.80 mmol/l) and moderate hypophosphatemia (10.9%, 0.32–0.65 mmol/l). The percentages of motile spermatozoa and progressively motile spermatozoa were lower in men with moderate hypophosphatemia than in men with mild hypophosphatemia or normophosphatemia (44%, 49%, 51%, P = 0.040, and 32%, 35%, 41%, P = 0.036, respectively). The total numbers of motile and progressively motile spermatozoa were also lower (13, 12, 18 million, P = 0.009, and 10, 9, 14 million, P = 0.006, respectively). Serum concentrations of total and free estradiol were highest in men with moderate hypophosphatemia (97.5, 96.2, 92.1 pmol/l, P = 0.004, and 2.4, 2.3, 2.2 pmol/l, P = 0.034, respectively). LIMITATIONS, REASONS FOR CAUTION The study question is compromised by the descriptive study design. It remains to be shown whether there exist a causal link between serum phosphate and semen quality in infertile men WIDER IMPLICATIONS OF THE FINDINGS As fertility stands as a critical concern in the world, there is a need to find regulators of fertility during adulthood to identify possible treatments. Therefore, the precise mechanisms through which hypophosphatemia may impact sperm motility remain needs to be further clarified. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by Novo Nordisk Foundation, Beckett Foundation, Medical doctor Sofus Carl Emil Friis and spouse Olga Doris Friis’s Grant, Candys Foundation, and The Innovation Foundation. There was no influence from any sponsor on the study design, and the authors have nothing to declare. TRIAL REGISTRATION NUMBER N/A.
... Many studies have shown that VD de ciency negatively affects semen quality and can be compensated by VD supplementation (21,37,49). Jensen et al. (50) stated that VDR level is a positive sign for sperm quality and reported that 1α,25-dihydroxyvitamin D3 may cause increased sperm motility by triggering VDR-mediated increase in intracellular Ca concentration in human spermatozoa. Sood et al. (51) reported a decrease in Sertoli cell function, degenerative changes in germinal epithelium, and a decrease in Leydig cells in rats with VD de ciency. ...
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Aim: In our study, we aimed to investigate the effects of VD against testicular damage caused by cisplatin by histopathological, immunohistochemical and biochemical methods. Material and Method: 28 rats were divided into four groups, control, VD, cisplatin and cisplatin+ VD groups. At the end of the 10-day experiment, the rats were sacrificed under ketamine/xylazine anesthesia. Right testicles were used for biochemical analyzes and left testicles were used for histological analyses. Sperm vitality and morphology were evaluated from semen samples obtained from the cauda part of the epididymis. Number of sperms was also counted. Biochemically, TOS, TAS, FSH, LH, testosterone, estrogen, VD, Ca and P levels were measured by ELISA test kits using spectrophotometric methods. Results: In histopathological analysis; a decrease in seminiferous tubule diameter and germinal epithelial thickness, decrease in Johnsen score, and degenerative changes in germinal cells were observed in the cisplatin group. While caspase-3 immune positivity increased, VDR immunostaining decreased. In biochemical analysis; while a significant increase was observed in TOS in the cisplatin group, no significant difference was found in terms of TAS. It was observed that VD application reduced histological damage and caused a significant increase in Johnsen score. In this group, caspase-3 immunostaining decreased while VDR immunostaining increased. Biochemically, a significant decrease in TOS level and a significant increase in VD level were detected. Conclusion: In conclusion, this study shows that VD administration alleviates testicular damage in rats with cisplatin-induced testicular damage. However, further studies are needed to add VD to clinical treatment protocols.
... The effects of vitamin D and calcium on oocyte development have important clinical implications for the success of in vitro fertilization (IVF). Vitamin D deficiency can lead to calcium deficiency, which results in disturbances in oocyte maturation, development, and fertilization [8][9][10] . ...
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Vitamin D and calcium in follicular fluid play an important role in modulating steroidogenesis, folliculogenesis, and oocyte quality determination. Both collaborate to produce top-quality embryos (TQE) during in vitro fertilization (IVF). In this study, we compared free 25(OH)D3 and calcium levels in follicular fluid between TQE and non-TQE groups. This cross-sectional study included women who underwent IVF procedures at tertiary hospitals in Bandung, Indonesia. Ovarian follicular fluid was collected during the ovum pick-up procedure. The examination of 25(OH)D3 levels, vitamin d-binding protein, and calcium in the follicles was done using an enzyme-linked immunosorbent assay (ELISA). Free 25(OH)D3 levels were calculated using the Vermeulen formula. A total of 173 samples met the study criteria, including 86 subjects in the TQE group and 87 subjects in the non-TQE group. There was a significant difference in free 25(OH)D3 follicular fluid levels between the TQE and non-TQE groups (p = 0.017); however, there was no significant difference in calcium levels between the two groups (p = 0.805). We also found that there was a significant association between free 25(OH)D3 follicular fluid levels and embryo quality (OR 3.05, 95% CI 1.46–6.38; p-value = 0.002); however, there was no significant association between follicular fluid calcium and embryo quality [p = 0.144 and OR, 1.74 (95% CI 0.82–3.68)]. The results suggest that free 25(OH)D3 and calcium in the follicular fluid act independently during steroidogenesis, folliculogenesis, and fertilization.
... Vitamin E and vitamin C have been shown to possess robust membrane protective capabilities [13]. Vitamin D has been shown to induce spermatozoon motility by increasing the intracellular calcium concentration [14]. Additionally, vitamin K has been demonstrated to regulate acrosin activity in spermatozoa [15]. ...
... Studies on the dual role of VitD in calcium homeostasis and gene expression have led to controversial findings regarding its impact on reproductive performance [7,8,9,10,11,12,13,28,29,30]. To differentiate between calciotropic and noncalciotropic effects, a well-established animal model of VDD was developed while maintaining normal calcium levels [13,14,16]. ...
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Vitamins A and D3 play essential roles in various physiological processes, including reproductive health. This study investigates the effects of Vitamin A and Vitamin D3 supplementation on sperm morphology in male subjects. The experimental design included a control group and two test groups, one receiving Vitamin A and the other Vitamin D3, over a 12-week period. Sperm samples were collected pre- and postsupplementation and analyzed for morphological changes using standardized criteria. Results indicate that both vitamins significantly improve sperm morphology, though through different mechanisms. Vitamin A supplementation was associated with enhanced acrosome integrity and overall sperm motility, while Vitamin D3 primarily contributed to increased sperm concentration and a reduction in morphological abnormalities. These findings suggest that adequate levels of Vitamins A and D3 are vital for optimal sperm health, potentially offering therapeutic avenues for treating male infertility.
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Context Cosmetic paraffin oil injections can lead to granuloma formation, causing hypercalcemia and kidney failure. Objective This study explores whether debulking surgery is an effective treatment for improving calcium homeostasis, inflammation, and clinical symptoms. Methods In a retrospective study, we reviewed 33 patients undergoing debulking surgery. Changes in calcium, inflammatory markers, and renal function from baseline up to 12 months after surgery were assessed. Patients were interviewed after surgery. Results The patients were 34.6 years of age (SD 6.9) and had 1104 grams (SD 591) of granuloma tissue removed following injection of 1329 mL (SD 803) paraffin oil 7.9 years (SD 3.2) earlier. Seventeen patients had hypercalcemia and experienced a significant decline in ionized calcium from 1.48 mmol/L (SD 0.16) at baseline to 1.33 mmol/L (SD 0.03) at 12 months (P < .002), although only 4 men (23.5%) became normocalcemic. Serum ferritin was reduced by 50% after 12 months (P = .048). Sixteen patients were normocalcemic and had no change in calcium homeostasis but experienced a 20% drop in serum ferritin levels (P = .025) after surgery. Fifteen patients completed all their planned surgeries within the study period and experienced a decline in serum ionized calcium (P = .031), ferritin (P = .011), and interleukin 2-receptor (P = .037). A survey showed that 55% of patients reported postoperative satisfaction scores of 10/10, and 59% of the patients reported reduced pain. Conclusion Surgery improved calcium homeostasis in a fraction of patients and reduced inflammation and subjective symptoms such as pain and mental well-being in a patient group left with few treatment options except high-dose prednisolone.
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The mitochondrial enzyme 25-hydroxyvitamin D 1α-hydroxylase, which is encoded by the CYP27B1 gene, converts 25OHD to the biological active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D). Renal 1α-hydroxylase activity is the principal determinant of the circulating 1,25(OH)2D concentration and enzyme activity is tightly regulated by several factors. Fibroblast growth factor-23 (FGF-23) decreases serum 1,25(OH)2D concentrations by suppressing CYP27B1 mRNA abundance in mice. In extra-renal tissues, 1α-hydroxylase is responsible for local 1,25(OH)2D synthesis, which has important paracrine actions, but whether FGF-23 regulates CYP27B1 gene expression in extra-renal tissues is unknown. We sought to determine whether FGF-23 regulates CYP27B1 transcription in the kidney and whether extra-renal tissues are target sites for FGF-23-induced suppression of CYP27B1. In HEK293 cells transfected with the human CYP27B1 promoter, FGF-23 suppressed promoter activity by 70%, and the suppressive effect was blocked by CI-1040, a specific inhibitor of extracellular signal regulated kinase 1/2. To examine CYP27B1 transcriptional activity in vivo, we crossed fgf-23 null mice with mice bearing the CYP27B1 promoter-driven luciferase transgene (1α-Luc). In the kidney of FGF-23 null/1α-Luc mice, CYP27B1 promoter activity was increased by 3-fold compared to that in wild-type/1α-Luc mice. Intraperitoneal injection of FGF-23 suppressed renal CYP27B1 promoter activity and protein expression by 26% and 60% respectively, and the suppressive effect was blocked by PD0325901, an ERK1/2 inhibitor. These findings provide evidence that FGF-23 suppresses CYP27B1 transcription in the kidney. Furthermore, we demonstrate that in FGF-23 null/1α-Luc mice, CYP27B1 promoter activity and mRNA abundance are increased in several extra-renal sites. In the heart of FGF-23 null/1α-Luc mice, CYP27B1 promoter activity and mRNA were 2- and 5-fold higher, respectively, than in control mice. We also observed a 3- to 10-fold increase in CYP27B1 mRNA abundance in the lung, spleen, aorta and testis of FGF-23 null/1α-Luc mice. Thus, we have identified novel extra-renal target sites for FGF-23-mediated regulation of CYP27B1.
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Article
Working Hypothesis Mutations in the CYP2R1 gene, highly expressed in the testis and encoding vitamin D 25-hydroxylase, result in a vitamin D deficiency and a defective calcium homeostasis leading to rickets. Objective Our aim was to investigate CYP2R1 expression in pathological testis samples and relate this to vitamin D metabolism in testiculopathic patients. Design, Patients, Setting Testis samples for in vitro study and 98 young men were transversally evaluated at Padova's Center for Male Gamete Cryopreservation. Methods CYP2R1 mRNA expression and protein production were evaluated by quantitative RT-PCR, Western blot analysis, and immunofluorescence. Hormonal and bone-marker levels, and bone densitometry by dual-energy x-ray absorptiometry, were determined in patients with Sertoli-cell-only syndrome and severe hypospermatogenesis. Results We found a lower gene and protein expression of CYP2R1 in samples with hypospermatogenesis and Sertoli-cell-only syndrome (P < 0.05) and a colocalization with INSL-3, a Leydig cell marker, at immunofluorescence. In all testiculopathic patients 25-hydroxyvitamin D levels were significantly lower and PTH levels higher compared to controls (P < 0.05). Furthermore, testiculopathic patients showed osteopenia and osteoporosis despite normal testosterone levels compared with controls both with increased bone-marker levels and altered dual-energy x-ray absorptiometry in the femoral neck and lumbar spine (for all parameters, P < 0.05). Conclusions Our data show an association between testiculopathy and alteration of the bone status, despite unvaried androgen and estrogen levels and no other evident cause of vitamin D reduction. Further studies in larger cohorts are needed to confirm our results.
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Vitamin D is known to reverse infertility in male and female rats. This study was an investigation of the effects of vitamin D deficiency on calbindin-D28K (CaBP28K) and testosterone levels in male chickens. Chickens were raised from 1 day of age to 8 wk of age on a normal or a vitamin D-deficient diet. A radioreceptor assay showed that serum vitamin D levels were significantly higher in chickens fed a normal diet than in those fed a vitamin D-deficient diet. The morphology of the seminiferous tubules was not different between the vitamin D-replete and vitamin D-deficient chickens. Immunohistochemical studies revealed that CaBP28K was present in spermatogonia and spermatocytes of the seminiferous tubules. A few interstitial Leydig cells were positive for CaBP28K. RIA was used to quantify the amount of CaBP28K in the testes, which was threefold higher in chickens raised on a normal diet than in chickens raised on a vitamin D-deficient diet. Testosterone concentration in serum, determined by RIA, was not different between the two groups. Neither serum calcium nor phosphorus levels were different between the two groups. This investigation represents the first demonstration of the effect of vitamin D deficiency on CaBP28K expression in chicken testes. The results indicate that the decrease in testicular CaBP28K concentration was attributable to vitamin D deficiency despite normal serum testosterone and calcium levels in 8-wk-old chickens.