BIOLOGY OF REPRODUCTION 84, 336–341 (2011)
Published online before print 29 September 2010.
DOI 10.1095/biolreprod.110.086157
Vitamin A Deficiency Results in Meiotic Failure and Accumulation of Undifferentiated
Spermatogonia in Prepubertal Mouse Testis1
Hui Li,3 Krzysztof Palczewski,5 Wolfgang Baehr,6 and Margaret Clagett-Dame2,3,4
Department of Biochemistry3 and Pharmaceutical Science Division,4 University of Wisconsin, Madison, Wisconsin
Department of Pharmacology,5 School of Medicine, Case Western Reserve University, Cleveland, Ohio
John A. Moran Eye Center,6 University of Utah Health Science Center, Salt Lake City, Utah
Vitamin A (retinol) is required for maintenance of adult
mammalian spermatogenesis. In adult rodents, vitamin A
withdrawal is followed by a loss of differentiated germ cells
within the seminiferous epithelium and disrupted spermatogenesis that can be restored by vitamin A replacement. However,
whether vitamin A plays a role in the differentiation and meiotic
initiation of germ cells during the first round of mouse
spermatogenesis is unknown. In the present study, we found
that vitamin A depletion markedly decreased testicular expression of the all-trans retinoic acid-responsive gene, Stra8, and
caused meiotic failure in prepubertal male mice lacking
lecithin:retinol acyltransferase (Lrat), encoding for the major
enzyme in liver responsible for the formation of retinyl esters.
Rather than undergoing normal differentiation, germ cells
accumulated in the testes of Lrat/ mice maintained on a
vitamin A-deficient diet. These results, together with our
previous observations that germ cells fail to enter meiosis and
remain undifferentiated in embryonic vitamin A-deficient
ovaries, support the hypothesis that vitamin A regulates the
initiation of meiosis I of both oogenesis and spermatogenesis in
mammals.
developmental biology, gamete biology, germ cells, male
reproductive tract, meiosis, retinoic acid, retinol, spermatogenesis, spermatogonia
INTRODUCTION
In the mouse embryo, male germ cells are enclosed by
Sertoli cells within the testis cords and arrest as G0/G1
spermatogonia. Within a few days after birth, male germ cells
resume mitosis and move toward the basement membrane of
the seminiferous cords, where they become undifferentiated
spermatogonia that initiate and maintain spermatogenesis. In
mice, spermatogenesis begins at the end of the first week of life
[1, 2] and continues throughout the lifetime of the adult male.
Spermatogenesis is a complex process involving mitotic
divisions, meiotic divisions, and dramatic morphological
changes ultimately resulting in spermatozoa. Undifferentiated
MATERIALS AND METHODS
Generation of VAD Prepubertal Mice
The Lrat/ mutant line has been described previously [8]. Female Lratþ/þ
and Lrat/ mice were maintained on a normal chow diet (LabDiet #5015;
TestDiet) up to approximately 8 wk of age. Female Lratþ/ þ mice were mated
with Lratþ/ þ males, and female Lrat/ mice were mated with Lrat/ males.
Mating females were examined daily for vaginal plugs, and the day when the
plug was found was considered to be Embryonic Day 0.5. At this time, the
females were placed on a purified diet devoid of vitamin A activity [12] and
supplemented daily with vitamins D2 (87.5 ng), E (77 lg of RRR-atocopherol), and K1 (3.5 lg) [13] without or with added retinyl palmitate (RP;
100 U/day) in soybean oil (VAD diet or vitamin A-sufficient [VAS] diet,
respectively). The resulting offspring remained with their mothers and
continued to receive the same diet until termination (P6, P10, or P18). A
subset of Lrat/ mothers on the VAD diet were supplemented with RP starting
at P5 (rescue experiment). All animal experimentation was reviewed and
approved by the University of Wisconsin-Madison College of Agricultural and
Life Sciences Animal Care and Use Committee.
1
Supported in part by grants EY008061 (K.P.) and EY019298 (W.B.)
from the National Institutes of Health (NIH).
2
Correspondence: Margaret Clagett-Dame, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison,
WI 53706-1544. FAX: 608 262 7122;
e-mail: [email protected]
Received: 20 May 2010.
First decision: 16 June 2010.
Accepted: 22 September 2010.
Ó 2011 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363
336
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spermatogonia divide to become differentiating spermatogonia
(type A1–4 spermatogonia, intermediate spermatogonia, and
type B spermatogonia), all of which are observed by Postnatal
Day (P) 8. Type B spermatogonia divide to form preleptotene
primary spermatocytes, which further develop into haploid
spermatids and spermatozoa [3].
The process of spermatogenesis is influenced by both
intrinsic and extrinsic factors, including vitamin A (retinol).
Vitamin A is required for the maintenance of mammalian
spermatogenesis, and most germ cells are lost in vitamin Adeficient (VAD) adult rodents. Vitamin A deficiency results in
spermatogenic arrest at the stage of undifferentiated spermatogonia and preleptotene spermatocytes in rats [4, 5] or type A
spermatogonia in mice [6], and retinol replacement results in
the complete recovery of spermatogenesis [4, 6, 7].
Despite increasing evidence indicating that vitamin A
regulates the initiation of meiosis of germ cells in the
developing ovary and maintains adult spermatogenesis, direct
evidence supporting a role for vitamin A in the initial
differentiation and meiotic entry of spermatogonia and, thus,
in the initiation of spermatogenesis in the prepubertal testis is
still lacking. This can be attributed largely to the lack of a
suitable animal model to induce vitamin A deficiency during
prepubertal life. Vitamin A is obtained from the maternal diet
and is stored primarily as a retinyl ester in the liver and, to a
lesser extent, in other tissues. Retinol is esterified primarily
through the action of the enzyme lecithin:retinol acyltransferase (LRAT) [8, 9], and Lrat/ mice are much more susceptible
to developing vitamin A deficiency than their wild-type
counterparts [10, 11]. In the present study, we use a nutritional
approach to produce vitamin A deficiency in the testis of
prepubertal Lrat/ mice before germ cells enter meiosis and
describe a role for vitamin A in spermatogonial differentiation
and meiotic initiation in these animals.
ABSTRACT
VITAMIN A NEEDED IN FIRST ROUND OF SPERMATOGENESIS
337
RNA Extraction and Quantitative RT-PCR Analysis
Freshly dissected gonads were snap-frozen in liquid nitrogen and stored at
808C. Total RNA was isolated and reverse transcribed as previously described
[14]. Quantitative PCR studies of Stra8 were performed using the Applied
Biosystems StepOnePlus Real-Time System with the Power SYBR Green PCR
Master Mix (Applied Biosystems). Values were normalized to 18S rRNA
expression levels. Values for Stra8 were expressed as a percentage of values
determined in samples from Lratþ/ þ mice maintained on the VAS diet. The
following primer sets were used: for Stra8, 5 0 -CTGTTGGACCAGATGCT
GAA-3 0 and 5 0 -GCAACAGAGTGGAGGAGGAG-3 0 ; for Rn18s, 5 0 -ACC
GCGGTTCTATTTTGTTG-3 0 and 5 0 -AGTCGGCATCGTTTATGGTC-3 0 .
PCR product quality was monitored by using post-PCR melting-curve analyses
at the end of the amplification cycles.
Immunofluorescence Studies
found in Lrat/ mice fed the VAS diet (Fig. 2). In contrast,
Stra8 levels were decreased by approximately 95% in Lrat/
mice maintained on the VAD diet as compared to the Lrat þ/ þ
VAS controls (Fig. 2). Thus, an increase in Stra8 mRNA was
not observed in the testes of VAD Lrat/ pups at P6, but an
increase was seen when these mice were supplemented with
dietary vitamin A. These results support the conclusion that
vitamin A deficiency was established by P6 in the testes of
Lrat/ mice from mothers fed the VAD diet.
Germ Cells in VAD Prepubertal Mouse Testes Fail
to Enter Meiosis
To assess the need for vitamin A for initiation of meiosis of
germ cells in the prepubertal testis, tissue sections were
prepared at P10 from Lrat þ/ þ or Lrat/ mice fed either the
VAS or VAD diet and were stained with hematoxylin. Germ
cells that had entered meiosis (as evidenced by the presence of
condensed chromosomes) were readily observed in the testes of
Lrat þ/ þ mice receiving either the VAS or VAD diet (Fig. 3, A
and B) as well as in Lrat/ mice receiving the VAS diet (Fig.
3C). In contrast, meiotic cells were rarely seen in the testes of
Lrat/ mice maintained on the VAD diet (Fig. 3D). Thus,
provision of dietary vitamin A prevented the meiotic failure
observed in VAD Lrat/ pups (Fig. 3, C and D).
We performed immunostaining for SYCP3 to verify these
findings. SYCP3 marks the thread-like synaptonemal complex
RESULTS
Adult Lrat/ Mice Given Dietary Vitamin A Show
No Apparent Defects in Male Germ Cell Development
To determine whether the Lrat gene is essential for male
germ cell maturation, testicular sections from 3-mo-old Lrat/
mice maintained on a standard VAS chow diet were stained
with hematoxylin. Lrat/ mice exhibited normal testicular
histology, with a full complement of maturing germ cells in
their seminiferous tubules (Fig. 1). Seven Lrat/ males bred to
23 Lrat/ females all yielded live pups, with an average litter
number of 8 6 1 pups, similar to the number of pups resulting
from 27 Lrat þ/ þ by Lrat þ/ þ crosses (7 6 1 pups/litter; mean 6
SD).
Stra8 Levels Are Decreased at P6 in Testes of Lrat/ Mice
Maintained on the VAD Diet
Stra8 is an all-trans retinoic acid (atRA)-responsive gene
[16] that is highly expressed in mouse testis beginning at Day 6
after birth [17], several days before the onset of meiosis I. Stra8
mRNA levels in the testes at P6 did not differ between Lrat þ/ þ
mice receiving either the VAS or VAD diet or from the levels
FIG. 2. Expression of Stra8 is reduced at P6 in the testes of Lrat/ mice
maintained on the VAD diet. Results are expressed relative to values
measured in the testes of Lratþ/ þ mice maintained on the control VAS diet,
arbitrarily set at one. Values are the mean 6 SEM of three independent
samples. *P , 0.01 vs. control using one-way ANOVA and Tukey post hoc
analyses.
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Dissected testes were fixed for 1 h in Bouin fixative (VWR Scientific
Products) and washed in PBS, dehydrated in 100% ethanol, embedded in
paraffin, and sectioned (thickness, 10 lm). Testes from three to four mice from
two to four independent litters were analyzed for each treatment group and time
point. For immunofluorescence studies, sections were dewaxed and boiled for
10 min in 10 mM citrate buffer (pH 6.0) in a microwave oven. Sections were
blocked in PBS containing 5% heat-inactivated serum (matched to the species
of the secondary antibody) and 0.1% Triton X-100 for 30 min at room
temperature before staining with primary antibodies. Sections were incubated
overnight at 48C with antibodies directed against DEAD box polypeptide 4
(DDX4, also known as MVH; 1:2000; kindly provided by Dr. Toshiaki Noce,
Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan), zinc finger and
BTB domain containing 16 (ZBTB16, also called PLZF; 1:200, 2A9;
Calbiochem), or synaptonemal complex protein 3 (SYCP3; 1:500; a kind gift
from Dr. Christa Heyting, Wegeningen University, Wegeningen, The Netherlands) [15]. After three washes in PBS, sections were incubated for 60 min at
room temperature with goat anti-rabbit Alexa 488 or goat anti-mouse Alexa
594 (Molecular Probes) diluted 1:500. After three additional washes in PBS,
slides were mounted with antifade medium (Biomeda) and analyzed with a
Nikon Eclipse TE 2000-U microscope. A negative control that omitted the
primary antiserum was included in each experiment.
For quantitative cell analyses, the number of DDX4- and ZBTB16-positive
cells per tubule in P6 testes were determined from more than 150 random
tubules from three to four mice for each treatment group. The percentage of
tubules containing SYCP3-positive cells in P10 testes was determined from
more than 150 random tubules from three to four mice for each treatment
group, and the total number of positive cells per field was determined from
three random fields per mouse testes. To determine the number of ZBTB16positive cells per tubule in P18 testes, ZBTB16-stained cells in more than 200
random tubules from four mice for each treatment group were counted.
Differences between the groups were evaluated using a one-way ANOVA and
the Tukey multiple-comparison test, with significance set at P , 0.05.
FIG. 1. Hematoxylin-stained sections of testes from 3-mo-old Lratþ/ þ (A)
and Lrat/ mice (B) show comparable germ cell development. Original
magnification 3600.
338
LI ET AL.
and the number per labeled tubule were also significantly lower
in Lrat/ mice on the VAD diet (Supplemental Fig. S1; all
Supplemental Data are available online at www.biolreprod.
org). Thus, in the prepubertal mouse testis, vitamin A is
required for the meiotic entry of germ cells during the first
round of spermatogenesis.
Male Germ Cell Development Is Unaltered at P6 in VAD
Lrat/ Pups
FIG. 3. Vitamin A deficiency results in meiotic failure of testicular germ
cells. Sections at P10 from testes of Lratþ/ þ mice fed the VAS (A and E) or
VAD (B and F) diet and Lrat/ mice fed the VAS (C and G) or VAD (D and
H) diet were stained with hematoxylin (A–D) or an antibody to SYCP3, a
structural component in the synaptonemal complex located along paired
meiotic chromosomes (E–H). Quantitation of the percentage of tubules
positive for SYCP3 antibody staining is also shown (I). *P , 0.05 vs. all
other groups. Original magnification 3200 (A–H) and 3600 (insets in A–
D).
found only in meiotic cells. Similar to the results obtained with
hematoxylin staining, SYCP3-labeled cells were found in 53%
and 52% of seminiferous tubules in Lrat þ/ þ mice that had
received the VAS or VAD diet, respectively, as well as in 55%
of tubules in Lrat/ mice that had received the VAS diet (Fig.
3, E–G and I). In contrast, SYCP3-positive cells were observed
in less than 1% of tubules in the testes of VAD Lrat/ mice
(Fig. 3, H and I). The number of SYCP3-labeled cells per field
To assess the effect of vitamin A deficiency on male germ
cell development, double-staining for DDX4 and ZBTB16 was
performed at P6 on testicular sections from Lrat þ/ þ or Lrat/
mice fed the VAS or VAD diet. DDX4 marks germ cells, and
ZBTB16 labels undifferentiated spermatogonia [18, 19]. The
number of germ cells and undifferentiated spermatogonia in
testes of Lrat/ mice maintained on the VAD diet did not
differ from the numbers in testes of Lrat þ/ þ mice maintained
on the VAS or VAD diet or in testes of Lrat/ mice
maintained on the VAS diet (Fig. 4 and Supplemental Fig. S2).
We next sought to determine whether germ cells in the VAD
testes at P6 still retained the ability to enter meiosis. Lrat/
mice were maintained on the VAD diet as described until P5, at
which time a daily supplement of RP was added to the diet.
Meiosis was rescued in the testis of these pups, as evidenced by
staining with both hematoxylin and SYCP3 (56% tubules
positive for SYCP3) (Fig. 5, A and C), whereas no meiosis was
evident in testes from Lrat/ mice not receiving supplemental
RP (Fig. 5, B and D). Thus, the developmental potential of
testicular germ cells and undifferentiated spermatogonia from
VAD Lrat/ pups at P6 appears to be unaltered, and
development can be rescued by the addition of dietary
vitamin A.
Germ Cells in VAD Testes Remain Undifferentiated
To determine whether the germ cells from VAD Lrat/
pups that failed to enter meiosis at P6 remained undifferentiated, immunostaining for ZBTB16, a marker of undifferenti-
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FIG. 4. Germ cell development appears unaltered at P6 in the VAD
Lrat/ testes. Germ cells were identified in the testes of Lratþ/ þ mice fed
the VAS (A) or VAD (B) diet and in sections from Lrat/ mice fed the VAS
(C) or VAD (D) diet by using antibodies to DDX4 (green; a marker for germ
cells) and ZBTB16 (red; a marker of undifferentiated spermatogonia).
Nuclei of cells (DNA) were labeled with 4 0 ,6 0 -diamidino-2-phenylindole
(blue). Original magnification 3200.
VITAMIN A NEEDED IN FIRST ROUND OF SPERMATOGENESIS
339
ated spermatogonia, was performed on testicular sections at
P18. In the control group in which Lrat þ/ þ mice were
maintained on a VAS diet, undifferentiated spermatogonia
lessened in number with age, corresponding to the time when
these cells undergo their expected differentiation (Fig. 6A).
Lrat þ/ þ mice on the VAD diet and Lrat/ mice on VAS diet
also showed a similar reduction in undifferentiated spermatogonia (Fig. 6, B and C). In contrast, the number of germ cells in
the VAD Lrat/ testes that remained as undifferentiated
spermatogonia (ZBTB16-positive) lining the basement membrane of the seminiferous tubule was significantly higher than
that in the other three groups (Fig. 6, D and E).
DISCUSSION
In the female mouse, meiotic entry of germ cells occurs
during embryogenesis, whereas in the male, the entry into
meiosis I occurs after birth and throughout adulthood.
Previously, we showed that vitamin A deficiency blocks the
initiation of meiosis of germ cells in developing ovaries [14].
Until now, it was not possible to determine whether vitamin A
is also required for the first meiotic division of the male germ
cell, because deficiency could not be induced before
spermatogenesis initiates at approximately P7. Using a genetic
mouse model that does not efficiently store vitamin A, we now
show that meiotic failure of germ cells occurs during the first
wave of male spermatogenesis.
Vitamin A deficiency is difficult to produce in wild-type
mice, because vitamin A is stored in the liver in the form of
retinyl esters [20] and neonates also receive vitamin A in
maternal milk [21]. LRAT is the predominant enzyme in the
liver responsible for the formation of retinyl esters [8–10], and
Lrat/ mice are more susceptible to developing vitamin A
deficiency than wild-type mice [10, 11]. In the present study,
vitamin A deficiency was produced in prepubertal life by
maintaining pregnant and lactating Lrat/ female mice on a
diet devoid of all forms of vitamin A. Generation of vitamin A
deficiency in the testes of pups at P6 was confirmed by
FIG. 6. Germ cells at P18 remain undifferentiated in the mouse VAD
Lrat/ testes. Sections from the testes of Lratþ/ þ mice fed the VAS (A) or
VAD (B) diet and from Lrat/ mice fed the VAS (C) or VAD (D) diet were
stained with an antibody to ZBTB16 (red); cell nuclei (DNA) were labeled
with 4 0 ,6 0 -diamidino-2-phenylindole (blue). Quantitation of the number
of ZBTB16 antibody-labeled cells per tubule is also shown (E). *P , 0.05
vs. all other groups. Original magnification 3200.
measuring the level of the atRA-responsive gene, Stra8.
Testicular expression of Stra8 is induced by atRA and serves
as an excellent marker for atRA activity [17, 22, 23]. Stra8
expression also is down-regulated by atRA deficiency in a
dose-dependent manner in the developing ovary [14]. In
normal mice, an increase in Stra8 first occurs in the testes at
P6, a few days before the onset of meiosis I. The highest Stra8
levels are reached by P10 [17], a time that coincides with the
onset of meiosis I. In Lrat/ pups maintained on the VAD
diet, the expected increase in Stra8 expression that is seen in
normal mice did not occur at P6 and was only 5% of the level
observed in wild-type mice fed the VAS diet, confirming the
establishment of atRA deficiency.
Germ cells fail to enter meiosis in the neonatal VAD Lrat/
testes at P10, as evidenced by the absence of condensed
chromosomes by histological analysis and the lack of SYCP3
expression, a marker found only in meiotic cells. In the adult
male, the need for vitamin A to maintain spermatogenesis is
well known. The adult VAD testis contains largely undifferentiated spermatogonia, and vitamin A is required for the
conversion of these cells to type A1 (differentiated) spermatogonia [24]. Treatment of cultured germline stem cells from P2
male mice with atRA produces an increase in Kit, a marker of
spermatogonial differentiation, suggesting that atRA plays a
normal role in this process in the neonate [25]. However,
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FIG. 5. Retinol supplementation prevents meiotic failure because of
vitamin A deficiency. Testicular sections from Lrat/ mouse pups
maintained on either the VAD diet from Embryonic Day (E) 0.5 to P5
followed by maternal supplementation from P5 to P10 (A and C) or the
VAD diet from E0.5 to P10 (B and D) were stained with hematoxylin (A
and B) or an antibody to SYCP3 (C and D). Original magnification 3200.
340
LI ET AL.
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evidence that vitamin A is required for meiotic initiation in the
neonatal testes in vivo was lacking. The present work proves
that vitamin A is required for meiotic initiation during the first
round of mouse spermatogenesis.
Meiotic failure in the prepubertal VAD testes cannot be
explained on the basis of altered germ cell numbers or impaired
ability to initiate the meiotic program. Expression of the germ
cell marker, DDX4, and of the undifferentiated spermatogonia
marker, ZBTB16, was unaffected by vitamin A deficiency,
showing that the number of germ cells in P6 VAD testes was
comparable to that in P6 VAS testes. Moreover, supplementation with retinol at P5 rescued the meiotic initiation. This is
consistent with our previous observation that germ cell
development appeared to be normal in the testis of VAD rat
embryos [14]. Taken together, these observations indicate that
vitamin A is not required for maintaining the undifferentiated
state of male germ cells during embryonic and prepubertal
development.
In both the male and female, meiotic failure is accompanied
by a marked decrease in Stra8 gene expression. In embryonic
ovarian germ cells of Stra8-null mutant mice, initiation of the
meiotic prophase is blocked, demonstrating that Stra8 gene
function is needed for meiotic entry in mouse embryonic
ovaries [26]. Unlike the situation in females, analysis of testes
of two Stra8-deficient lines of mice revealed less definitive
results. In one Stra8-deficient mouse line, germ cells failed to
enter meiosis in the prepubertal testis [27], whereas analysis of
a second Stra8-deficient line showed that male germ cells can
enter meiosis but fail to complete this process [28]. Therefore,
whether meiotic failure in prepubertal VAD testes is a direct
result of the absence of Stra8 gene expression in these atRAdeficient germ cells requires further study.
Interestingly, spermatogonia in the prepubertal VAD testes
that fail to enter meiosis remain in an undifferentiated state as
the pups continue to develop. In the normal P18 testes, germ
cells at various stages of spermatogenesis (spermatogonia,
primary and secondary spermatocytes, and round spermatids)
are present, and the number of undifferentiated spermatogonia
gradually declines with the progression of spermatogenesis [1].
In contrast, in the VAD testes at P18, a large number of
undifferentiated spermatogonia were found, and they accumulated at the basement membrane of seminiferous tubules.
Interestingly, in ovaries of VAD rat embryos, germ cells that
fail to undergo meiosis remain undifferentiated [14], similar to
the undifferentiated spermatogonia in prepubertal VAD testes,
and CYP26B1 maintains low levels of retinoic acid in
embryonic testes to block meiotic initiation of germ cells [23,
29, 30]. It appears, therefore, that an environment depleted of
retinoic acid maintains germ cells in an undifferentiated state in
both sexes. Thus, the VAD Lrat/ mouse testis provides an
enriched source of undifferentiated spermatogonia that may be
useful for future studies concerning the regulation of
spermatogonial differentiation.
In summary, the present study shows that vitamin A is
needed for spermatogonia to enter meiosis I during the first
wave of spermatogenesis in prepubertal mice. Spermatogonia
remain in an undifferentiated state when vitamin A is absent,
resulting in an accumulation of undifferentiated spermatogonia.
These results support the hypothesis that vitamin A and its
derivative, atRA, regulate the initiation of meiosis I during
mammalian spermatogenesis.
VITAMIN A NEEDED IN FIRST ROUND OF SPERMATOGENESIS
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