ISRAEL JOURNAL OF
VETERINARY MEDICINE
Vol. 57 (2) 2002
VITAMIN D RECEPTORS IN VITAMIN D - REPLETED
AND - DEPLETED CHICKEN TESTES, AND THEIR
EFFECTS ON DEVELOPMENT
I. Kurtul
Department of Anatomy, Faculty of Veterinary Medicine, Ankara
University, Ankara, Turkey
Abstract
Occupied nuclear and cytosolic testicular vitamin D receptors (VDR), were identified in the
testes of various animal species. This study shows the localization of VDRs in the
seminiferous tubules of vitamin D-repleted and - depleted chicken testes. White leghorn
male chicks were raised from 1 day to 12 weeks of age in vitamin D-repleted and depleted groups. Chickens were weighed before they were euthanized, and testes were
weighed after removal. Testes were fixed and cut serially for immunohistochemistry.
Immunohistochemical staining indicated specific staining for VDRs in spermatogonia,
spermatocytes and Sertoli cells from vitamin D-repleted chickens. By contrast, the same
cells from vitamin D-depleted chicken testes showed either faint staining or none at all.
These results reveal the effect of vitamin D on germ cell development, and show that
vitamin D exerts its action via VDRs in the testes of chickens.
Introduction
Recent studies on additional target tissues for vitamin D, include the parathyroid gland,
pancreas, ovary, testis, skin, placenta, brain, liver, prostate and parotid gland (1,2,3,4).
Vitamin D receptors (VDR), in these new target tissues has been identified by the use of
increasingly sensitive biochemical assays for nuclear 1,25-dihydroxyvitamin D3 receptors.
Since the detection of VDR in reproductive tissues of various species, its action has been a
broad research subject. Unoccupied nuclear and cytosolic VDRs have been shown in some
chick tissues including intestinal mucosa, and in male rat tissues, including the testes (5). The
occupied nuclear and cytosolic testicular VDRs in the seminiferous tubules of adult rodents
have been identified (6). This study showed that VDRs were present in Sertoli cells; but were
absent in Leydig cells.
The nuclei of Sertoli cells in the testes and epithelium of the ductuli efferentes have been
shown to be targets for vitamin D3 in the mouse, implying that at a molecular level, vitamin D
playes a role in reproduction (7). In an in vitro study using adult rats, a specific but lowcapacity binding agent for vitamin D3 was found in isolated seminiferous tubules and
interstitial tissue of adult rats; however, no specific binding of VDR was detected in immature
Sertoli cell (8). The testicular VDR was shown to be present in Sertoli cells, being highest at
the stage of spermiogenesis in the mouse (9). The extensive presence of binding sites for
vitamin D3 was detected in Sertoli cells and the caput epididymidis at a time of spermiosis in
the testes, suggesting that vitamin D is involved in a certain process during spermatogenesis
and in sperm maturation in rats (8,10). The concentration of VDR changes throughout
development from being undetectable in immature rats but rapidly increasing at maturity (11).
This study showed that the increase was due to an augmentation in the number of VDR sites
and not due to a change in the affinity of the receptors.
Another study of vitamin D-deficient rats showed that there was incomplete spermatogenesis
and degenerative changes in the testes, along with some evidence that the testicular VDRs
were located in Sertoli cells (12). These changes suggest that the Sertoli cell is a primary site
of action for vitamin D3. Since VDRs are mainly located in Sertoli cells, it has been
speculated that these receptors function in germ cell division and maturation, possibly by an
indirect effect via Sertoli cells. In a study using Siberian hamsters, it was concluded that
vitamin D3, probably regulates the effect of follicle stimulating hormone (FSH) on Sertoli cell
function and testicular growth (13). The effects and interaction of sex hormones and vitamin D
metabolites on alkaline phosphatase were shown in rat endometrial cells in primary culture
(14).
In the most recent study, immunohistochemical localization and tissue distribution of VDRs in
male and female rat reproductive tissues have been demonstrated by the use of primary
antibodies against VDR (15). In the study, VDRs have been shown in spermatogonia,
spermatocytes, Sertoli cells, epithelial cells of the epididymis, prostate and seminal vesicles of
male rats. The results from this study revealed that Leydig cells and fibroblast cells of the
testis did not show immunostaining for VDR. Since, both nuclear and cytoplasmic receptor
staining were shown in male rat reproductive tissues, VDR was thought likely to mediate the
actions of vitamin D3 in these tissues.
The aim of this study is to locate VDR in the testes of vitaminD-depleted and -repleted
chickens, and to define its effects on them.
Materials and Methods
Tissue preparation: White leghorn male chicks were raised from 1 day to 12 weeks of age in
vitamin D-depleted and -repleted groups. The groups were fed vitamin D-depleted diet + 3%
calcium and vitamin D-repleted diet + 3% calcium. The diets had previously been analyzed for
of vitamin-D and Ca levels. There were 9 chickens in each group. Chickens were weighed
before they were euthanized with sodium pentobarbital. Testes were also weighed after
removal.
Fixation and Immunohistochemical Location: Testes were fixed by indicated method (16).
Fixed tissues were processed and embedded in paraffin by described techniques (17,18).
Five-micron sections were serially cut. Tissue sections of the testes of the groups were first
deparaffinized and incubated with 5% urea, 5% normal goat serum (NGS) in phosphate
buffer solution (PBS), to gain a low background, rat anti-vitamin D receptor in 1: 200 dilution
in 1% of NGS in PBS overnight, respectively. The sections were also incubated with
nonimmune serum in the same manner. They were then incubated with biotinylated goat antirat IgG diluted 1:100 in PBS only, peroxidase labeled streptavidin diluted 1:100 in 1% NGS in
PBS, and 0.05% 3,3’-diaminobenzidine hydrochloride, DAB, & 0.01 % hydrogen peroxide in
0.05 M tris buffer. Tissue sections of mature mouse testes were used as positive controls.
Results
Imunohistochemical staining indicated specific staining for VDR in the seminiferous tubules,
specifically in spermatogonia, spermatocytes and Sertoli cells from vitamin D-repleted
chickens (Fig. 1 A, B). Adjacent sections incubated with nonimmune serum, normal rat serum,
are also shown in Figure 4. Seminiferous tubules of the vitamin D-repleted groups that
received an adequate amount of vitamin D were stained intensely. Nuclear immunostaining of
VDR was intense in spermatocytes and spermatogonia, and less so in Sertoli cells. On the
other hand, the seminiferous tubules of the testes of vitamin D-depleted chickens showed
either faint staining or none at all (Fig. 2 A, B).
Seminiferous tubules of mature mouse testes were used as a positive control for VDR since
these were previously shown to possess VDR (7). Specific immunostaining was found in the
seminiferous tubules especially in spermatogonia, spermatocytes and Sertoli cells of the
mature mouse testes (Fig. 3).
Figure 1. Testes from vitamin D-repleted chicken, demonstrating VDR immunoreactivity in
the spermatogonia, spermatocytes and Sertoli cells (arrows).
A) X200, B)X400.
A
B
Figure 2. Testes from vitamin D-depleted chicken. Immunoreactivity for VDR is eirher faint or
absent in the spermatogonia, spermatocytes and Sertoli cells (arrows).
A) X200, B)X400.
A
B
Figure 3. Testes from mature mouse used as a positive control, showing VDR
immunoreactivity in the spermatogonia, spermatocytes and Sertoli cells (arrows).
X400.
Figure 4. Immunoreactivity of section adjacent to that in figure 1, stauned with normal
serum.
X200.
Discussion
With the development of very sensitive biochemical assays and immunohistochemistry,
VDRs have been found in many unexpected tissues (2,3,4). The testes of many species were
shown to be a target for vitamin D (8,9,13,15).
In this study, immunohistochemical staining showed specific staining for VDR in the
seminiferous tubules, specifically in spermatogonia, spermatocytes and Sertoli cells. Specific
staining for VDR in the seminiferous tubules of the vitamin D-depleted chicken testes was
either faint or not detected. This result indicates that vitamin D modulates its receptor in the
nucleus of target cells by feedback control.
Immunohistochemical staining was highest when spermatogenesis occurred, suggesting the
possible involvement of vitamin D in sperm maturation (9). Increasing levels of VDR as the
animal matures also implies the possible involvement of vitamin D in sperm maturation. The
increase in VDR during development of the testes showed that it was due to an increase in
the number of receptor sites, and not an increase in the affinity of the receptors (11). In this
study, since the Sertoli cell was identified as the main target of vitamin D, it is suggested that
vitamin D is involved in the process of sperm cell nourishment by the Sertoli cell. The testes
of vitamin D-depleted animals have shown incomplete spermatogenesis, impaired
development, and degenerative changes in the seminiferous tubules. As seen in these
studies , it has been clearly indicated that function of vitamin D is probably related to germ
cell division and maturation in the testis. Since the Sertoli cell is thought to be the main target
for vitamin D, the function of vitamin D in the testes may be an indirect effect via the Sertoli
cells.
Vitamin D, probably after the second hydroxylation to 1,25 D3 in the kidney, was shown to
modulate the effects of follicle-stimulating hormone on Sertoli cell function and testicular
growth in Siberian hamsters (13). In this study it was speculated that vitamin D3 could be
involved in the process of increasing the number of FSH receptors in the testes, and/or it
could increase the sensitivity of Sertoli cells to FSH stimulation. VDR are present in the caput
epididymis where the process of water and mineral resorption takes place. This study also
suggested vitamin D has a role in this process. The presence of VDR in smooth muscles of
the epididymis and deferent duct also suggests a possible role for vitamin D in sperm
transport.
In conclusion, VDRs are present in the seminiferous tubules especially in spermatogonia,
Sertoli cells and spermatocytes in vitamin D-repleted chicken testes. In vitamin D-depleted
chickens, development of the seminiferous tubules was impaired, and spermatogenesis was
disrupted. The increasing level of VDR through maturation indicates the importance of vitamin
D in reproduction. The results in this study suggest that vitamin D plays a very important
role(s) in the maturation of germ cells. Over all, it can be concluded that vitamin D is involved
in reproduction, presumably at the molecular level.
LINKS TO OTHER ARTICLES IN THIS ISSUE
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