BIOLOGY OF REPRODUCTION 78, 537–545 (2008)
Published online before print 21 November 2007.
DOI 10.1095/biolreprod.107.064337
Expression of Stimulated by Retinoic Acid Gene 8 (Stra8) and Maturation of Murine
Gonocytes and Spermatogonia Induced by Retinoic Acid In Vitro1
Qing Zhou,3 Ying Li,3 Rong Nie,3 Patrick Friel,3 Debra Mitchell,3 Ryan M. Evanoff,3 Derek Pouchnik,3
Brent Banasik,3 John R. McCarrey,4 Christopher Small,3 and Michael D. Griswold2,3
School of Molecular Biosciences,3 Washington State University, Pullman, Washington 99164
Department of Biology,4 University of Texas, San Antonio, Texas 78249
ABSTRACT
Vitamin A deficiency in the mouse results in an arrest in the
progression of undifferentiated spermatogonia to differentiating
spermatogonia. The supplement of retinol to vitamin-A-deficient
mice reinitiates spermatogenesis in a synchronous manner
throughout the testes. It is unclear whether the effects of
retinoids are the result of a direct action on germ cells or are
indirectly mediated through Sertoli cells. The expression of
Stimulated by retinoic acid gene 8 (Stra8), which is required for
spermatogenesis, is directly related to the availability of retinoic
acid (RA). Analysis of gene expression by microarrays revealed
moderate levels of Stra8 transcript in gonocytes and high levels
in A and B spermatogonia. Stra8 mRNA levels were greatly
reduced or absent in germ cells once they entered meiosis. This
study examined the effect of retinoic acid on cultured neonatal
testes and isolated gonocytes/spermatogonia in vitro. THY1+ and
KIT+ germ cells were isolated by magnetic-activated cell sorting
from the testes of mice of different ages. Isolated germ cells were
cultured and treated with either vehicle (ethanol) or RA without
feeder cells. We found that 1) Stra8 is predominantly expressed
in premeiotic germ cells, 2) RA stimulates gonocyte DNA
replication and differentiation in cultured neonatal testes, 3) in
the absence of feeder cells, RA directly induces the transition of
undifferentiated spermatogonia to differentiating spermatogonia
by stimulating Stra8 and Kit gene expression, 4) RA dramatically
stimulates Stra8 expression in undifferentiated spermatogonia
but has a lesser impact in differentiating spermatogonia, 5)
endogenous Stra8 gene expression is higher in differentiating
spermatogonia than in undifferentiated spermatogonia and
could mediate the RA effects on spermatogonial maturation,
and 6) RA stimulates a group of genes involved in the
metabolism, storage, transport, and signaling of retinoids.
differentiation, gonocytes, in vitro, retinoic acid, spermatogenesis,
spermatogonia, spermatogonial differentiation, Stra8
INTRODUCTION
In many adult male mammals, sperm production is a
continuous process in which millions of sperm are generated
each day, throughout life. Sperm production is truly one of the
1
Supported by NIH grants R01 HD 10808 and U54 HD 042454. The
microarray data have been deposited in the National Center for
Biotechnology Information Gene Expression Omnibus database (series
no. GSE9521).
2
Correspondence: Michael D. Griswold, 531 Fulmer Hall, School of
Molecular Biosciences, Washington State University, Pullman, WA
99164-4660. FAX: 509 335 9688; e-mail: mgriswold@wsu.edu
Received: 17 July 2007.
First decision: 10 August 2007.
Accepted: 2 November 2007.
Ó 2008 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
537
most dynamic cell-producing systems in the body, and it
depends on the active proliferation and differentiation of
spermatogonial stem cells (SSC) [1]. SSC originate from
primordial germ cells, which, in turn, become gonocytes once
they migrate to the genital ridges and become enclosed by
differentiating Sertoli cells [2]. In rodents, gonocytes divide for
a few days and then arrest in the G0/G1 phase of the cell cycle.
The gonocytes arrested in mitosis migrate from the center of
the cords to the peripheral region and reach the basement
membrane. In mice, this migration is completed within the first
few days after birth, and the gonocytes resume proliferation
and gonocytes/prespermatogonia differentiate into SSC in the
same period [1, 2].
In rodents, SSC are single cells located on the basement
membrane of the seminiferous tubules and are called A-single
(As) spermatogonia. The SSC divide either into two new single
cells or into a pair of spermatogonia (Apr) that do not complete
cytokinesis and stay connected by an intercellular bridge. The
Apr spermatogonia divide further to form chains of 4, 8, and
occasionally, up to 32 A-aligned (Aal) spermatogonia. The Aal
spermatogonia go through a differentiation step and become A1
spermatogonia [3]. In rodents, there are five divisions
following A1 formation, forming successively A2, A3, A4, In
(intermediate), and B spermatogonia. The B spermatogonia
divide into spermatocytes. Each step of differentiation except
the transition of Aal to A1 is associated with a mitotic division.
As spermatogonia are considered the only true SSC, even
though A s, A pr, and A al are termed ‘‘undifferentiated
spermatogonia,’’ whereas spermatogonia from A1 to B are
termed ‘‘differentiating spermatogonia’’ [3].
The massive production of mature sperm is dependent on
successive mitotic divisions and associated cell differentiation
of spermatogonia. A few factors involved in the regulation of
spermatogonial differentiation have been identified [4],
including retinoic acid (RA) [5, 6], cyclin D2 (CCND2) [7],
KIT/KITL [8, 9], and DAZL [10]. It has been known since
1925 that vitamin A is required for normal spermatogenesis.
Spermatogenesis ceases and only A spermatogonia are found in
vitamin-A-deficient (VAD) mouse testes. Supplementing
vitamin A (retinol) to VAD mice reinitiates spermatogenesis
in a synchronous fashion throughout the testis [11]. Haneji et
al. [12] showed that both retinol and RA could induce
differentiation of type A spermatogonia in cultured mouse
cryptorchid testes. This conclusion was based on the
morphological identification of In and type B spermatogonia
after 9 days of culture with retinoids [12]. However, it is not
known whether the action of RA in inducing spermatogonial
differentiation is a direct effect on germ cells or an indirect
effect mediated through Sertoli cells, because both spermato-
gonia and Sertoli cells possess nuclear receptors for retinoids
[2, 13]. Moreover, the molecular mechanism of RA action in
spermatogonia is obscure. In a recent study, RA was
538 ZHOU ET AL.
demonstrated to induce the expression of Stra8, a factor
essential for the onset of meiosis in both male and female
gonads [14]. The knockout of the Stra8 gene blocks entry into
meiosis in both embryonic ovaries and pubertal testes [14, 15].
Thus, the expression of Stra8 appears to be a sensitive indicator
of the availability of RA and entry of cells into meiosis.
Several different methods to collect spermatogonia enriched
in SSC have been established, with one of the most effective
ways being magnetic-activated cell sorting (MACS) directed
against cell surface markers [16]. THY1 is likely a common
surface marker for all undifferentiated spermatogonia, and
THY1-positive selection using MACS results in significant
enrichment of SSC [16]. This technique allows the examination
of factors that potentially regulate the proliferation and
differentiation of spermatogonia in a controlled, in vitro
system [17]. Our study utilized short-term cultured neonatal
mouse testes and specific MACS subpopulations of spermato-
gonia using Stra8 and Kit as endpoints to test two objectives: 1)
determine if RA can induce the in vitro differentiation of early
germ cells in the absence of somatic cells and 2) identify the
subpopulation of spermatogonia in which RA induces Stra8
expression and spermatogonial differentiation.
MATERIALS AND METHODS
Animal Care and Treatment
All animal experiments were approved by Washington State University
Animal Care and Use Committees and were conducted in accordance with the
guiding principles for the care and use of research animals of the National
Institutes of Health. A BL/6–129 mouse colony was maintained in a
temperature- and humidity-controlled room with food and water provided ad
libitum. To investigate the induction of STRA8 protein expression by RA in
neonatal gonocytes, 2-day-postpartum (dpp) male mice were injected
subcutaneously with all-trans-RA (90% oil, 10% ethanol) at a dose of 17.5
lg/mouse or injected with vehicle as control. Testes were fixed 24 h later in
freshly prepared 4% (w/v) paraformaldehyde for immunohistochemical staining
of STRA8 protein. Four mice were used in each treatment group.
Testicular Cell Types and RNA Preparation
Enriched germ cell isolates for microarray analysis were prepared via
gravity sedimentation from CD1 mice as previously described [18]. These
samples included type A and B spermatogonia, pachytene spermatocytes, and
round spermatids from animals 8, 30, and 60 days of age, respectively. The
purity of all the isolated and cultured cells was determined by morphology. The
purity of type A and B spermatogonia was .85%, and the purity of all other
germ cell types was .95%. The quality control of the total RNA samples was
performed as previously described [19]. Two replicates were used in microarray
analysis.
Microarray Processing
The following method was used for microarray processing of 2 dpp/5 dpp
gonocytes, type A/type B spermatogonia, pachytene spermatocytes, round
spermatids, and Kit-deficient (W/Wv) mutant testes. Ten micrograms of total
RNA from each sample was used to create the target for the microarray. The
labeled cRNA was hybridized to Mouse Genome 430 2.0 arrays (Affymetrix,
Santa Clara, CA) and stained in accordance with the manufacturer’s standard
protocol. The same procedure was performed on duplicate samples. The arrays
were stained utilizing the Affymetrix GeneChip Fluidics Station 400 and
scanned using a GeneArray Scanner 2500A (Agilent Technologies, Palo Alto,
CA). The resulting data were viewed and analyzed using GeneSpring software
(Agilent Technologies, Santa Clara, CA). All reactions and microarray
hybridization procedures were performed in the Laboratory for Biotechnology
and Bioanalysis I at Washington State University. The different cell type data
was normalized within GeneSpring using the default/recommended normali-
zation methods. These include setting of signal values below 0.01 to 0.01, total
chip normalization to the 50th percentile. These normalizations allowed for the
visualization of data based on relative abundance at any cell type or organ
rather than compared to a specific control value.
A different method was used for microarray analysis of RA-treated 2 dpp
THY1þ gonocytes. Approximately 75 ng total RNA from each sample was
used to generate cDNA using the Ovation RNA Amplification System V2
(NuGEN, San Carlos, CA) according to the manufacturer’s instructions. Array
quality was assessed, and images were quantified using GeneChip Operating
Software v1.2 (Affymetrix). The data were viewed and analyzed using
GeneSpring software. The criteria for RA-regulated genes included a raw signal
value of no less than 50 in either control or treated samples, a 2-fold or higher
change in signal between the control and treated samples, and the consistent
changes in duplicates.
Neonatal Testis Organ Culture and Treatments
Neonatal testes from 2 dpp mice were detunicated, cut into four to six small
pieces per testis, placed on Millicell CM filters (Millipore, Bedford, MA)
floating on the surface of medium and covered with drops of medium
(Dulbecco modified Eagle medium/F12/10% fetal bovine serum) and cultured
for 24 h. The cultured testes were treated with all-trans-RA (ATRA) or vehicle
(ethanol). The final concentration of ATRA, 9 cis-RA, 4-oxo-RA, and retinol
acetate (ROL) in the culture medium was 0.7 lM [14], and the final
concentration of ketoconazole was 0.94 lM. After 24 h, tissues were harvested,
and RNA was isolated using the PicoPure system (Arcturus Bioscience,
Mountain View, CA). For studies involving RNA, three vehicle-treated and
three ATRA-treated cultured testes were used. For studies of 5-bromo-2 0 -
deoxyuridine (BrdU) incorporation, the vehicle control group contained five
testes, and the RA-treated group contained six cultured testes. The final
concentration of BrdU in the culture medium was 10 lM. The testis tissues
were fixed in Bouin solution after treatment.
Immunohistochemical Double Staining of BrdU and Germ
Cell Nuclear Antigen 1
Tissues were fixed in Bouin solution and embedded in paraffin. Tissues
were dewaxed and incubated in 2N HCl at 378C for 30 min. The staining
procedure was performed as previously described, with minor modifications
[19]. Briefly, mouse BrdU antibody (1:200 dilution, Vision BioSystems,
Norwell, MA) was applied first and incubated at 48C overnight. After three
washes in PBS, tissues were incubated with secondary antibody (tetramethyl-
rhodamine-isothiocyanate-conjugated donkey anti-mouse IgG, 1:100; Jackson
ImunoResearch Laboratory, West Grove, PA) at 378C for 1 h. Tissues were
blocked with 10% donkey serum and incubated with rat germ cell nuclear
antigen 1 (GCNA1) antibody (1:50 dilution; gift from Dr. George Enders of the
University of Kansas) at 378C for 2 h, and then secondary antibody (1:100
dilution; fluorescein-isothiocyanate-conjugated donkey anti-rat IgG; Jackson
ImunoResearch Laboratory, West Grove, PA) was applied at 378C for 1 h.
Sections incubated in PBS without primary antibody were used as negative
controls for color development on the same slide. Images were captured with an
Olympus OLY-200 digital camera (Olympus America, Inc., Melville, NY)
using Olympus MagnaFire Camera Imaging and Control (version 1.0; Olympus
America) and compiled using Adobe PhotoShop 7.0 (Adobe Systems, San
Jose, CA). The number of GCNA1-positive cells in each testis section was
counted first. The GCNA1 and BrdU staining images were overlapped in
PhotoShop, and double positive cells were counted. Finally, the percentage of
BrdU-positive germ cells per total germ cell number was calculated. The
Student t-test was used for statistical analysis, and P , 0.05 was considered to
be statistically significant.
Production of Anti-STRA8 Serum
To generate STRA8 antibody, a peptide termed P3 and corresponding to the
C-terminus (C L N Q E P E P P D D) of mouse STRA8 protein was synthesized
and conjugated with carrier protein maleimide activated keyhole limpet
hemocyanin (mcKLH, Pierce, Rockford, IL) following manufacturer’s
instructions. At Day 0, preimmune serum was collected from a New Zealand
rabbit, then 1 mg of conjugated protein in 0.7 ml of PBS mixed with 0.7 ml of
Freund complete adjuvant was injected into the rabbit subcutaneously. At Day
28, the rabbit was given a booster of 0.5 mg conjugated protein in Freund
incomplete adjuvant. The STRA8 antiserum was harvested 2 wk after the
booster injection.
Western Blot Analysis, Immunohistochemistry, and
Antibody Competition
The specificity of the STRA8 antibody was tested in three ways: a Western
blot analysis using P19 cells, immunohistochemistry performed on normal
adult mouse testes and on Stra8-deficient mouse testes, and antibody
 RA-INDUCED IN VITRO DIFFERENTIATION OF GONOCYTES
competition with P3 peptide (corresponding to the C-terminus of STRA8
protein and used in STRA8 antibody generation) in Western blot and
immunohistochemistry. Briefly, P3 peptide (300 lg/ml) was incubated together
with STRA8 antibody overnight at 48C, then the mixture was used as primary
antibody as described later. Protein from the murine embryonic carcinoma cell
line P19 cells (ATCC, Manassas, VA) was used for Western blot analysis
because Stra8 was first identified in this cell line [20]. P19 cells were cultured
based on the instructions of ATCC and were treated with either vehicle ethanol
or 1 lM RA (final concentration in the culture medium) for 24 h. Protein was
harvested in 23 Laemmli buffer and then separated by 12% SDS-PAGE and
transferred to nitrocellulose membrane (0.45 lm). The membrane was blocked
in PBS containing 5% nonfat milk and 0.025% Tween 20 for 1 h at room
temperature and incubated with STRA8 antiserum (1:500) overnight at 48C.
Total proteins from MSC1 cells (a Sertoli cell line) were used as negative
control. After incubation, membranes were rinsed with PBS with Tween 20 and
incubated with horseradish peroxidase conjugated goat anti-rabbit IgG for 1 h
at room temperature. Reactive proteins were visualized and exposed to films by
Western lighting chemiluminescence reagent (Perkin Elmer, Boston, MA). For
immunohistochemistry, serial sections of the mouse testis incubated with
preimmune serum (1:500) were used as negative controls.
Immunohistochemistry of STRA8 Protein in Neonatal Testes
Neonatal testes were removed 24 h after RA injection and fixed in freshly
prepared 4% (w/v) paraformaldehyde. Antigen retrieval in 0.01 M sodium
citrate, pH 6.0, was done using microwave radiation and a trypsin (1 mg/ml)
digestion for 7 min at 378C. For immunohistochemical staining, ZYMED LAB-
SA SYSTEM (Zymed Laboratory, South San Francisco, CA) was used,
following the manufacturer’s instructions. Briefly, tissue sections were
incubated with 10% goat serum followed by incubation with STRA8 antiserum
(1:2000) or preim (1:2000) at 48C overnight. The slides were incubated with
diluted biotinylated secondary antibody for 45 min, followed by the application
of streptavidin-horseradish peroxidase. The diaminobenzidine solution was
applied until brown color developed. Staining was observed with a Nikon
Microphot-FX (Meridian Instrument Company, Kent, WA) microscope.
Photographs were taken with an Olympus OLY-200 digital camera using
Olympus MagnaFire Camera Imaging and Control version 1.0 (Olympus
America).
Real-Time RT-PCR
A two-step real-time RT-PCR was used to measure the expression of
candidate genes as previously described [19]. Each RNA sample was analyzed
in triplicate with primers for each gene. Stra8 primers amplify a 151-bp product
(primers: 5 0 -GTTTCCTGCGTGTTCCACAAG-3 0 and 5 0 -CACCCGAGGCT-
CAAGCTTC-3 0 ); control Rps2 primers amplify a 112-bp product (primers: 5 0 -
CTGACTCCCGACCTCTGGAAA-3 0 and 5 0 -GAGCCTGGGTCCTCTGAA-
CA-3 0 ); Kit primers amplify a 100-bp product (primers: 5 0 -GATGGCG-
TTCCTCGCCT-3 0 and 5 0 -GCCCGAAATCGCAAATCTTT-3 0 ). Expression of
Stra8 and Kit were normalized to Rps2 expression. The Student t-test was used
to analyze the results from neonatal testes, and a pair-wise t-test was used to
analyze the results of all experiments using isolated cells. Data represent mean
6 SEM.
MACS of Gonocytes/Spermatogonia
Testes were collected from 2 dpp, 4 dpp, or 8 dpp mouse pups into Hanks
balanced salt solution, and the tunica were removed. MicroBeads with mouse
CD117 (KIT) and CD90 (THY1) antibodies and MS columns (Miltenyi Biotec,
539
FIG. 1. Stra8 mRNA expression in differ-
ent germ cell types by microarray analysis.
Values represent raw signal of Stra8 6 SEM.
2 dpp, 2 d post partum gonocytes; 5 dpp, 5
d post partum gonocytes; Type A, type A
spermatogonia; Type B, type B spermato-
gonia; Pachy, pachytene spermatocytes;
Round, round spermatids; W/Wv, adult
W/Wv mutant testes.
Auburn, CA) were used in the isolation according to manufacturer’s
instructions. The culture was performed based on Kubota and Brinster’s
method with modifications [16]. For experiments using 8 dpp testes, the cells
were always subjected to THY1þ MACS first, then the THY1– cells were
subjected to KITþ MACS to obtain two separate cell populations. Isolated cells
were cultured under feeder-cell-free, serum-free, and growth-factor-free
conditions for 8 or 24 h with only vehicle (ethanol) or ATRA (0.7 lM final
concentration) or ROL (0.7 lM final concentration). For each cell isolation,
twelve to fifteen 2 dpp, ten to twelve 4 dpp, and six to ten 8 dpp mice were used
to ensure a high enough number of cells for the subsequent experiments. Each
treatment group contained at least three independent repeats.
STA-PUT Gonocyte Isolation
Testes from 2 dpp mice were enzymatically digested as described by
O’Brien et al. [21] with modifications. Gonocytes or spermatogonia were
identified by phase contrast microscopy and pooled. The identity of the cells
was confirmed by GCNA1 immunocytochemistry, and 80%–90% of the cells
obtained by STA-PUT were GCNA1 positive (data not shown).
Immunocytochemistry of POU5F1 (OCT4) and GCNA1
Spermatogonia isolated from 8 dpp testes by MACS (THY1 or KIT
positive) were fixed in 4% paraformaldehyde (with 0.25% glutaraldehyde) for
POU5F1 staining and in Bouin solution for GCNA1 staining. After a few PBS
washes, cells were incubated with 3% H2O2 for 20 min at room temperature
and then blocked with 10% normal goat serum (for POU5F1) or 10% normal
rabbit serum (for GCNA1) for 30 min (goat anti-rabbit IgG for POU5F1
staining, Zymed; rabbit anti-rat IgM for GCNA1 staining, Cortex Biochem, San
Leandro, CA). After PBS washes, primary antibodies, either rabbit polyclonal
anti-POU5F1 (1:100; Abcam, Cambridge, MA) or GCNA1 (1:50), were added
and incubated at 378C for 2 h, followed by a PBS rinse and biotin-conjugated
secondary antibody incubation at 378C for 1 h. Diaminobenzidine was used as
substrate for peroxidase after streptavidin conjugation. The cells subjected to
GCNA1 staining were counterstained with hematoxylin (Sigma-Aldrich, St.
Louis, MO), and cells stained for POU5F1 were counterstained with nuclear
fast red (Vector, Burlingame, CA).
RESULTS
Stra8 Expression in Several Types of Premeiotic Germ Cells
The gene expression profile of Stra8 mRNA was obtained
by using microarray results from isolated germ cell samples
(Fig. 1). Stra8 mRNA was expressed in 2 dpp and 5 dpp
gonocytes, type A spermatogonia, and type B spermatogonia.
The expression level of Stra8 mRNA decreased significantly in
meiotic and postmeiotic germ cells with the lowest levels of
expression observed in pachytene spermatocytes and round
spermatids. In the adult W/Wv mutant testis, Stra8 expression
was at background levels.
RA Stimulation of DNA Replication in Vitamin-A-Sufficient
Neonatal Gonocytes
It has been shown that retinoids can induce DNA replication
and proliferation in arrested A spermatogonia in VAD adult
540 ZHOU ET AL.
FIG. 2. Immunohistochemical double
staining of GCNA1 and BrdU in cultured
neonatal testes. A) Immunostaining of
GCNA1 (green). B) Immunostaining of BrdU
(red). C) 4 0 ,6-diamidino-2-phenylindole
counterstaining of cell nuclei (blue). D)
Overlay of images A, B, and C. Nuclei of
proliferating germ cells appear yellow.
Original magnification 3100 (A–D) and
3200 (inset).
rats [5]. The effect of RA on gonocytes/prespermatogonia in
vitamin-A-sufficient, normal neonatal testes was examined.
Neonatal testes (2 dpp) were cultured for 24 h with or without
RA, fixed in Bouin solution, and embedded in paraffin. Tissue
sections were double-stained with GCNA1 and BrdU antibod-
ies (Fig. 2). The BrdU-positive germ cells were counted and the
percentage of BrdU-positive gonocytes/spermatogonia per total
germ cell number calculated. The percentage of BrdU-positive
gonocytes/spermatogonia per total germ cell number increased
17% (44% 6 6% vs. 61% 6 5%; P , 0.01) in the testes
treated with RA, compared to vehicle-treated testes.
RA Induction of Stra8 and Kit mRNA Expression in Cultured
Vitamin-A-Sufficient Neonatal Testes
Previous studies demonstrated that males and females share
an identical meiotic initiation pathway in which RA induces
Stra8 expression in both embryonic ovaries and adult testes,
and Stra8 is required for the transition into meiosis in both
male and female germ cells [14, 15]. The action of RA on the
expression of Stra8 mRNA in 2 dpp testes was examined in
organ culture by real-time RT-PCR. ROL (0.7 lM), RA (0.7
lM), and 9-cis-RA (0.7 lM), one major retinoic acid
metabolite 4-oxo-RA and a pan-P450 enzyme inhibitor
ketoconazole (0.9 lM) were utilized in this study (Fig. 3A).
In 2 dpp mouse testes cultured on floating membranes, RA
significantly increased the expression of Stra8 17-fold 24 hr
after treatment (P , 0.01), and 9-cis-RA treatment increased
Stra8 expression 8-fold (P , 0.05). Retinol acetate and 4-oxo-
RA increased Stra8 expression 7.8-fold and 8.7-fold, respec-
tively. Ketoconazole interferes with the degradation of RA by
inhibiting the enzymatic activity of CYP26B1 [14]. Ketocona-
zole alone did not make a significant change in Stra8
expression. However, ketoconazole seems to reduce the
magnitude of the effect of RA. KIT has been demonstrated
to be a marker for differentiating spermatogonia [8, 9]. We
found that the expression of Kit transcript was increased 4-fold
on average (P , 0.05) after RA treatment for 24 h (Fig. 3B).
RA Induction of STRA8 Protein Expression in Vitamin-A-
Sufficient Neonatal Testes
Western blot analysis confirmed that the STRA8 antibody
recognized a 44–46 Kd protein product in P19 cells with or
without RA treatment (Fig. 4, lanes 3 and 4) and did not detect
any signal in MSC1 cells (Fig. 4, lane 1) as expected. The
STRA8 band was completely abolished when antibody was
incubated overnight with P3 peptide (Fig. 4, lanes 6 and 7),
which was used to generate this antibody. STRA8 protein
expression was mostly undetectable in the gonocytes of 2 dpp
(starting age) testes treated by oil vehicle (for 24 h) by
immunohistochemistry with only 0.03% of the tubules on
average containing STRA8-positive germ cells (Fig. 5A).
However, in 24 h RA-treated 2 dpp testes, STRA8 protein was
induced in the cytoplasm of some, but not all, gonocytes, and
the percentage of tubules containing STRA8-positive germ
cells increased to 41% (P , 0.05; n ¼ 4; Fig. 5B). Preimmune
serum was used as negative control and did not show any
detectable positive staining. The positive immunostaining of
STRA8 in spermatogonia in testes of 2 dpp mice was abolished
by incubating antibody with P3 peptide overnight (data not
shown). No immunohistochemical staining was detected using
this antibody on testes from Stra8-deficient mice [15] (data not
shown).
RA Induction of Stra8 Expression in Cultured Gonocytes/
Prespermatogonia Without Feeder Cells
Previous studies have shown that RA can induce Stra8
expression in cultured embryonic testes and ovaries and also in
VAD adult testes [14]. Whether this RA induction is a direct
effect on germ cells or an indirect effect mediated through
testicular somatic cells, in particular Sertoli cells, is still
unknown. Neonatal gonocytes were isolated by THY1-positive
selection from 2 dpp and 4 dpp testes. THY1 antigen is
considered to be a surface marker for male germline stem cells,
and THY1-positive selection was used extensively to isolate
 RA-INDUCED IN VITRO DIFFERENTIATION OF GONOCYTES
spermatogonial stem cells for germ cell transplantation
experiments [16, 22]. The purity of the cell population was
examined by immunocytochemical staining of GCNA1, and
approximately 90% of the cells used in the following
experiments were GCNA1 positive. The isolated THY1 þ
gonocytes/spermatogonia were cultured under feeder-cell-free,
serum-free, and growth-factor-free conditions and treated with
retinoids for 8 h. In THY1þ gonocytes isolated from 2 dpp
testes, RA and ROL increased the Stra8 expression level 31-
fold (P , 0.01) and 17-fold (P , 0.01), respectively (Fig. 6A).
In THY1 þ gonocytes/spermatogonia isolated from 4 dpp testes,
RA and ROL stimulated Stra8 expression 20-fold (P , 0.01)
and 16-fold (P , 0.01), respectively (Fig. 6B). Before
establishment of THY1 sorting methods, STA-PUT sedimen-
tation was used to separate different types of germ cells, based
on their sizes and shapes [18]. To test whether gonocytes/
prespermatogonia isolated by STA-PUT would confirm the RA
induction of Stra8 expression observed in THY1-positive cells,
isolated gonocytes (85%–90% purity as confirmed by GCNA1
staining) from 2 dpp testes were subjected to identical RA/ROL
treatments. Stra8 mRNA expression increased 47-fold by RA
(P , 0.05) and 25-fold by ROL in the 8-h treatment (Fig. 6C).
RA Induction of Stra8 Expression in Undifferentiated and
Differentiating Spermatogonia
Premeiotic male germ cells include primordial germ cells,
embryonic gonocytes, prespermatogonia in neonatal testes,
spermatogonia, and preleptotene spermatocytes. In the sper-
matogonial population, As, Apr, Aal (undifferentiated sper-
matogonia), and differentiating spermatogonia, including A1–4,
AIn, and B, have been described and studied [3]. We
investigated whether RA could induce Stra8 expression in
spermatogonia, and, if so, in which specific cell type does RA
induce Stra8 expression? Eight-day-old mouse testes contain
undifferentiated and differentiating spermatogonia. KIT is a
surface protein preferentially expressed in differentiating
541
FIG. 3. Induction of Stra8/Kit expression
by retinoids in cultured neonatal testes
examined by real-time RT-PCR. Culture/
treatment duration is 24 h. Values represent
relative fold change of Stra8 or Kit 6 SEM.
A) Induction of Stra8 expression by different
types of retinoids and pan-P450 inhibitor
ketoconazole in cultured 2 dpp testes. B)
Induction of Kit expression by RA in
cultured 2 dpp testes. Asterisk (*) indicates a
statistically significant difference (P , 0.05)
between the labeled sample and control
sample. C, vehicle control (ethanol); 9-CIS,
9-cis-RA; 4-OXO, 4-oxo-RA; KETO, keto-
conazole.
spermatogonia [8]. THY1-positive sorting has been used to
isolate cells enriched with SSC [22], which we believe is
preferentially expressed in undifferentiated spermatogonia.
Therefore, we isolated THY1þ and KIT þ spermatogonial cells
from 8 dpp testes. The isolated cells were subjected to the
THY1 þ and KIT þ MACS sequentially to minimize potential
variation. POU5F1 (previously known as OCT4) was used as a
marker for undifferentiated spermatogonia [23]. Using immu-
nocytochemical staining for GCNA1 and POU5F1, we found
that 80%–90% of the spermatogonial cells isolated from 8 dpp
testes by THY1 þ selection were GCNA1 þ/POU5F1 þ, imply-
ing that the majority of these cells are undifferentiated
spermatogonia. Meanwhile, 40%–50% of the KIT þ spermato-
gonial cells isolated by this means were also POU5F1 positive
and classified as differentiating spermatogonia. The expression
of Stra8 was shown to be 15-fold higher (P , 0.05) in
differentiating (KIT þ) spermatogonia than in undifferentiated
(THY1 þ) spermatogonia. In order to determine if there was a
FIG. 4. Western blot analyses and STRA8 antibody. Western blot results
of STRA8 (against P3 peptide) antibody with total proteins from P19 cells
and MSC1 cells before and after antibody competition with P3 peptide.
Lanes 1, 2, 3, and 4 were exposed to STRA8 antibody only, and lanes 6, 7,
and 8 were exposed to anti-STRA8 serum preincubated with P3 peptide.
Lane 1: MSC1; lane 2: prestained molecular weight marker 1; lane 3: P19
þ vehicle (ethanol); lane 4: P19 þ RA; lane 5: prestained molecular
weight marker 2; lane 6: P19 þ RA; lane 7: P19 þ vehicle; lane 8: MSC1.
542 ZHOU ET AL.

FIG. 5. Immunohistochemical detection of STRA8 protein induced by RA in cultured neonatal testes. A) Immunostaining of STRA8 after 24 h oil-treated
(starting at age 2 dpp mouse testis). B) Immunostaining of STRA8 after 24 h RA treated (starting at age 2 dpp mouse testis). Inset shows a dividing STRA8-
positive gonocyte (arrow). Original magnification 3200 (A, B) and 3400 (inset).

difference between undifferentiated and differentiating sper-
matogonia with respect to their abilities to respond to RA
stimulation, the isolated spermatogonia were cultured for 8 h in
the absence of feeder cells with or without RA. Stra8
expression was increased 15.7-fold (P , 0.05) in THY1 þ
spermatogonia in 8 h, whereas there was only a 2.7-fold
increase in Stra8 expression in KIT þ spermatogonia (Fig. 7).
RA Induction of Cell Differentiation in Cultured
Undifferentiated Spermatogonia
It has been shown that retinoids induce differentiation of
arrested A spermatogonia in VAD rat testes and cultured
cryptorchid testes. It is unknown whether retinoids can induce
the differentiation of murine As, Apr, or Aal spermatogonia in
the absence of somatic cells in vitro. To this end, the
expression of Kit after RA treatment under culture conditions
without feeder cells was examined. Consistent, but not
statistically significant, up-regulation of Kit after 8 h of RA
treatment was observed in THY1 þ spermatogonial cells (data
not shown). Because the S phase of A spermatogonia lasts
about 25 h, and differentiation (after a mitotic division) into A2
spermatogonia occurred only 48 h after retinoid stimulation in
vivo under VAD conditions [5], the RA treatment duration was
extended to 24 h, and 3.6-fold up-regulation (P , 0.05) of Kit
expression in THY1 þ spermatogonial cells was detected.
Altered Expression of Genes Involved in Vitamin A
Metabolism and Signaling in RA-treated 2 dpp THY1 þ
Gonocytes Revealed by Microarray Profiling
Using the restrictions as described in Materials and
Methods, a signal level of at least 50 in at least one of the
samples and the expression levels being consistently up- or
down-regulated by 2-fold in the two duplicates, 80 transcripts
were up-regulated, and 97 transcripts were down-regulated by
RA in 8 h in THY1þ gonocytes. A group of genes involved in
the transport, storage, metabolism, and signaling of retinoids
was significantly up-regulated by RA stimulation, including
Stra6, Lrat, Cyp26a1, Cyp26b1, Por, Dhrs3, Rarb, and Stra8
(Table 1). A complete list of the transcripts will be available
through the Griswold Lab home page (http://www.wsu.edu/

FIG. 6. Induction of Stra8 expression by RA/ROL (8 h) in cultured neonatal gonocytes without feeder cells examined by real-time RT-PCR. Values
represent relative fold change of Stra8 expression 6 SEM. A) Induction of Stra8 expression by RA/ROL in THY1þ 2 dpp gonocytes cultured without feeder
cells for 8 h. B) Induction of Stra8 expression by RA/ROL in THY1þ 4 dpp gonocytes cultured without feeder cells for 8 h. C) Induction of Stra8 expression
by RA/ROL in 2 dpp gonocytes isolated by STA-PUT and cultured without feeder cells for 8 h. Culture medium did not contain serum or any growth factor.
Asterisk (*) indicates a statistically significant difference (P , 0.05) between the labeled sample and control sample. C, vehicle control.
 RA-INDUCED IN VITRO DIFFERENTIATION OF GONOCYTES
FIG. 7. RA induction of Stra8 expression in THYþ and KIT þ spermato-
gonia in 8 h without feeder cells. Values represent relative fold change of
Stra8 expression 6 SEM. Asterisk (*) indicates a statistically significant
difference (P , 0.05) between the RA-treated sample and control sample.
THY1þ, THY1þ spermatogonia; KIT þ, KITþ spermatogonia.
griswold/microarray) and the National Center for Biotechnol-
ogy Information Gene Expression Omnibus (http://www.ncbi.
nlm.nih.gov/projects/geo/query/acc.cgi?acc¼GSE9521).
DISCUSSION
In this study we found that 1) Stra8 was predominantly, if
not exclusively, expressed in premeiotic germ cells, 2) RA
artificially induced Stra8 and Kit expression and increased
gonocyte DNA replication in cultured 2 dpp testes, and 3) RA
induced STRA8 protein in the cytoplasm of some, but not all of
the 2 dpp gonocytes, 4) RA can directly stimulate Stra8
expression in vitro in premeiotic male germ cells without
support from somatic cells, 5) RA induced expression of the
spermatogonial differentiation marker gene, Kit, in the same
feeder-cell-free culture environment, 6) RA dramatically
stimulated Stra8 expression in undifferentiated spermatogonia
but had a lesser impact on differentiating spermatogonia, 7) the
endogenous level of Stra8 expression is higher in differenti-
ating than in undifferentiated spermatogonia, and 8) RA
significantly stimulated the expression of a group of genes
involved in the metabolism, storage, transport, and signaling of
retinoids.
Based on these findings, a detectable level of gonocyte/
spermatogonia differentiation can be achieved in vitro by RA
stimulation in the absence of somatic support. In addition,
Stra8 is very likely a critical component mediating the RA
regulation of spermatogonial differentiation. Previous studies
suggest that, in the development of early spermatogonia, RA
induces the transition of either Aal to A1 or A1 to A2
spermatogonia [5, 11]. RA induction of Stra8 expression is
probably a vitally important trigger to the subsequent
TABLE 1. Transcripts involved in retinoid metabolism, storage, transport and signaling that are up-regulated by RA greater than 2-fold in 2 dpp THY1þ
gonocytes.
Genes GenBank accession no. Fold change
Lrat BB518473
Cyp26a1 NM_007811
Stra8 NM_009292
Rarb BB266455
Dhrs3 NM_011303
Stra6 NM_009291
Cyp26b1 AW049789
Por NM_008898
543
differentiation steps and commitment toward the formation of
primary spermatocytes. It is worth noting that different types of
prespermatogonia, including embryonic gonocytes and early
neonatal gonocytes, were capable of responding to RA
induction as well [14].
The recent findings from several studies revealed that RA is
the signal triggering the onset of meiosis in embryonic ovaries
[14, 24], and Stra8 is the downstream factor required for this
commitment [14]. In the study of Koubova et al. [14], the same
regulatory pathway was also confirmed in adult VAD testes. In
the male germline, a series of developmental events occur
within the first 10 days after birth. The male germ cells do not
enter meiosis until 8–10 dpp, as evidenced by the first
appearance of preleptotene spermatocytes. This current study
demonstrates RA induction of Stra8 mRNA and STRA8
protein in cultured neonatal testes prior to meiotic initiation,
which suggests that the first round of meiosis very likely shares
the same mechanisms in embryonic ovaries and adult testes.
Further, the strong induction of DNA replication by RA
indicates that RA might promote the differentiation of these
early gonocytes, because almost every step of spermatogonial
development is associated with a mitotic division [3].
However, the possibility that RA also stimulates the self-
renewal of these gonocytes cannot be ruled out. RA induction
of Kit expression in these neonatal testes ultimately substan-
tiates the hypothesis that RA triggers the differentiation of 2
dpp gonocytes, because KIT is proven to be a marker for
differentiating spermatogonia.
Several lines of evidence suggest that neonatal gonocytes
represent a heterogeneous population. In the early 1970s,
Zamboni and Merchant [25] observed bridges connecting some
of the gonocytes, indicating that these cells had already
differentiated. Later, de Rooij [1, 2] proposed that at the
beginning of spermatogenesis some gonocytes give rise to SSC
(As), and others directly differentiate to A1 spermatogonia. A
more recent result by Yoshida et al. [23], analyzing the unique
expression of Ngn3, strongly indicates that the first round of
spermatogenesis initiates directly from gonocytes, and they
suggest that the state of undifferentiated spermatogonia is not
an inevitable step but a developmental option. These recent
data show that if an appropriate signal is given, the direct
transition of gonocytes to A1 spermatogonia can be induced in
an in vitro culture system. This report suggests that the signal
inducing differentiation is RA. In this study, RA induced
STRA8 protein in some of the 2 dpp gonocytes but not in all of
them. This is a strong indication of heterogeneity of neonatal
gonocytes. It is possible that some of these neonatal gonocytes
could directly enter the phase of differentiating spermatogonia,
and others would commit to becoming SSC. Koubova et al.
[14] demonstrated that RA could artificially induce Stra8 in
embryonic testes. However, the period of incubation of the
embryonic testes was 2 days, and the possibility of serial,
accelerated differentiation events, including the conversion of
Function
12 Retinoids absorption and storage
11 Metabolism of RA
9 Onset of meiosis
9 Nuclear receptor of RA
6 Metabolism of all-trans-retinal
5 Membrane receptor for retinol binding protein
3 Metabolism of RA
2 Metabolism of RA
544 ZHOU ET AL.
embryonic gonocytes to undifferentiated spermatogonia occur-
ring after RA stimulation, could not be excluded.
The results from cultured neonatal testes generated two
major observations. First, in culture systems with intact somatic
support, RA induces Kit expression, a marker of differentiating
spermatogonia. Second, it also induces Stra8, a marker for the
onset of meiosis. These two observations question whether
somatic cells are required for RA induction of gonocyte
differentiation in vitro. Development of a method to induce in
vitro differentiation of SSC has been widely pursued, and some
studies show some progression of spermatogenesis in a culture
system. However, the starting materials used in previous
reports are mostly preleptotene or leptotene spermatocytes [26–
28]. Our study is the first report of spermatogonial differen-
tiation induced by one extrinsic factor in vitro. RA is a factor
that can induce differentiation in many organs and cells. It has
been demonstrated that RA could induce the differentiation of
Aal to A1 in VAD animals and cultured cryptorchid adult testes
[5, 12]. However, whether the RA effect on early germ cells is
direct or indirect has not been determined, because receptors
for retinoids are found in both germ cells and somatic cells.
Retinoids play pivotal regulatory roles in Sertoli and Leydig
cells and also interstitial macrophages. Our results obtained
from cultured THY1þ gonocytes/spermatogonia in a feeder-
cell-free condition demonstrate, for the first time, that in vitro
differentiation of undifferentiated early germ cells could be
initiated by RA without somatic cells and that the differenti-
ation of these cells can be validated by the induction of
expression of two markers, Kit and Stra8. Whether the
differentiation triggered by RA in this somatic-free system
can lead to formation of late differentiating spermatogonia (In
and B spermatogonia) and primary spermatocytes is not clear,
because germ cells under this feeder-cell-free and serum-free
condition could survive only up to 48 to 72 hours (data not
shown). It is noteworthy that in both cultured THY1 þ
gonocytes and neonatal testes, RA-stimulated germ cells
showed a higher percentage of cell death after the first 24 h
(data not shown). This strongly suggests that once RA triggers
the differentiation of these germ cells, the somatic cells,
particularly the mature Sertoli cells, become vital to their
survival and are likely to be important for the continuing
progression of the differentiation pathway.
If Stra8 is required for the onset of male meiosis, when does
RA act during spermatogonial development and which
subpopulation of spermatogonia is responsible for Stra8
expression? Analysis of Stra8-deficient testes demonstrated
that Stra8 is essential for normal progression into meiotic
prophase [14, 15]. The findings of robust induction of Stra8/Kit
expression after 8 h using 2 dpp, 4 dpp, and 8 dpp THY1 þ
gonocytes/spermatogonia suggest that induction occurs during
gonocyte/spermatogonial maturation. There is additional evi-
dence supporting this conclusion: 1) previous studies from de
Rooij and others [3] suggest that some gonocytes directly
differentiate to A1 in the first round of spermatogenesis and 2)
because the first appearance of KIT was found in late Aal to A1,
and it was confirmed to be a marker of differentiating
spermatogonia (A1-B), the magnitude and temporal sequences
of induction of Stra8 vs. Kit expression in THY1 þ gonocytes
implies that RA induction of Stra8 expression might be earlier
than induction of Kit expression. So far, data demonstrate that
RA initially induces Stra8 expression, and gonocyte and
spermatogonia differentiation occurs. However, some ques-
tions arise from this study that prevent the exclusion of the
possibility of a second induction at B-preleptotene-leptotene.
For instance, which subpopulation of differentiating spermato-
gonia (late Aal, A1, or B spermatogonia) contributes to the
higher level of Stra8 in KIT þ cells?
Microarray profiling of RA-treated 2 dpp THY1þ gonocytes
not only confirmed the induction of Stra8 expression
demonstrated by real-time RT-PCR in this study, but also
revealed that a group of genes involved in multiple aspects of
RA action were significantly up-regulated by RA. The
induction of Stra6, which was originally identified as an RA
responsive gene in P19 carcinoma cells [29], further validates
the effectiveness of RA treatment on these gonocytes.
Recently, STRA6 was found to be a specific membrane
receptor of retinol binding protein, representing a major
physiological mediator of cellular vitamin A uptake [30]. So
far, these gonocytes are the only known noncancer cells to
stimulate Stra6 expression in response to RA. The increased
Stra6 expression indicates a facilitated uptake of retinol into
the cells, and the physiological relevance of this up-regulation
in gonocytes needs to be studied. In the meantime, an
accelerated RA degradation in these gonocytes upon the RA
treatment was indicated by the dramatic increase of expression
of three genes: Cyp26a1, Cyp26b1, and Por. CYP26A1 and
CYP26B1 are two of the three P450 enzymes in the CYP26
subfamily to oxidize RA for further metabolism and inactiva-
tion [31]. Cytochrome P450 oxidoreductase (POR) coded by
Por is an NADPH-dependent protein essential for transferring
electron to microsomal cytochrome P450 [31]. Lack of POR
would impair the activity of all three CYP26 enzymes [31]. In
addition to the increased metabolism of RA, the synthesis of
RA is likely attenuated, as indicated by the induction of Lrat
and Dhrs3. Lecithin-retinol acyltransferase (LRAT) is the
predominant enzyme in the body responsible for the physio-
logic esterification of retinol [32]. Increased Lrat indicates a
potentially increased storage of retinyl ester and reduction of
supply of retinol for the production of RA. Dehydrogenase/
reductase member 3 (DHRS3) regenerates retinol from all-
trans-retinal, which is an intermediate in the production of all-
trans-RA [33]. The increased DHRS3 could mean a potential
reduction of retinal for the synthesis of RA. Other RA-
regulated genes in the gonocytes are being analyzed and
studied. The data obtained from this microarray experiment
will provide a fertile basis for the hypothesis-driven studies of
RA-regulated gonocyte differentiation.
In conclusion, this study demonstrates that a limited level of
in vitro differentiation of gonocytes/spermatogonia could be
achieved after RA stimulation without somatic involvement. In
addition, RA induction of Stra8 expression in the undifferen-
tiated spermatogonia was demonstrated, implying its important
role in spermatogonial differentiation.
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