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Dev Biol. Author manuscript; available in PMC 2018 December 15.
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Published in final edited form as:

Dev Biol. 2017 December 15; 432(2): 229–236. doi:10.1016/j.ydbio.2017.10.002.

Retinoic acid deficiency leads to an increase in spermatogonial

stem number in the neonatal mouse testis, but excess retinoic
acid results in no change
Kellie S. Agrimsona,1, Melissa J. Oatleya, Debra Mitchella, Jon M. Oatleya, Michael D.
Griswolda, and Cathryn A. Hogartha
aSchool of Molecular Biosciences and the Center for Reproductive Biology, Washington State
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University, Pullman, Washington, USA

Abstract
The onset of spermatogenesis occurs in response to retinoic acid (RA), the active metabolite of
vitamin A. However, whether RA plays any role during establishment of the spermatogonial stem
cell (SSC) pool is unknown. Because designation of the SSC population and the onset of RA
signaling in the testis that induces differentiation have similar timing, this study asked whether RA
influenced SSC establishment. Whole mount immunofluorescence and flow cytometric analysis
using the Id4-eGfp transgenic reporter mouse line revealed an enrichment for ID4-EGFP+ cells
within the testis following inhibition of RA Synthesis by WIN 18,446 treatment. Transplantation
analyses confirmed a significant increase in the number of SSCs in testes from RA-deficient
animals. Conversely, no difference in the ID4-EGFP+ population or change in SSC number were
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detected following exposure to an excess of RA. Collectively, reduced RA altered the number of
SSCs present in the neonatal testis but precocious RA exposure in the neonatal testis did not,
suggesting that RA deficiency causes a greater proportion of progenitor undifferentiated
spermatogonia to retain their SSC state past the age when the pool is thought to be determined.

Keywords
retinoic acid; spermatogenesis; testis; spermatogonial stem cell

Introduction
Within the neonatal mouse testis, establishment of the spermatogenic lineage initiates when
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prospermatogonia, the earliest germ cell precursors present at birth, migrate to the basement
membrane of the seminiferous cord. After migration, these cells follow one of two fates: 1)
transition directly into differentiating spermatogonia to produce the first round of

Correspondence: Cathryn A. Hogarth, School of Molecular Biosciences, Washington State University, Pullman, WA 99164.
chogarth@wsu.edu.
1Present address: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota, USA
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 Agrimson et al. Page 2

spermatogenesis, or 2) become undifferentiated spermatogonia. However, whether the

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undifferentiated population is solely SSCs or both SSCs and committed progenitors is

unknown. Progenitor spermatogonia are known to contain the cell machinery to respond to
RA signaling and proceed through the differentiation process (de Rooij and Russell, 2000),
yet there is no published evidence to indicate that the SSCs can respond to RA. The best
predictor of stem cell or progenitor capacity during steady state spermatogenesis is inhibitor
of DNA binding 4 (ID4) levels, particularly when trying to detect the transitional state
during which SSCs transition into progenitors (Chan et al., 2014; Helsel et al., 2017). ID4 is
a helix-loop-helix protein that binds other helix-loop-helix transcription factors, acting as a
repressor (Benezra et al., 1990; Riechmann et al., 1994). Transplantation from transgenic
Id4-eGfp mice demonstrated that the ID4-EGFP+ spermatogonial population contains most
of the SSC population (Chan et al., 2014; Helsel et al., 2017). Further analyses of ID4-EGFP
+ spermatogonia have revealed that the intensity of the EGFP signal correlates with
regenerative capacity, with the ID4-GFPBright population being pure SSCs (Helsel et al.,
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2017).

Previous work suggested that the SSC pool forms between 0 and 6 dpp (Bellve et al., 1977;
Chan et al., 2014; de Rooij and Russell, 2000; Huckins and Clermont, 1968; McLean et al.,
2003). This timing correlates with the onset of RA signaling in the testis that occurs between
3 and 5 dpp (Busada et al., 2014; Snyder et al., 2011; Snyder et al., 2010). RA deficiency,
induced by diet, genetically, or chemically using the retinaldehyde dehydrogenase inhibitor
WIN 18,446, has been shown to block spermatogonial differentiation, producing testes
lacking advanced germ cells but enriched with undifferentiated spermatogonia (Griswold et
al., 1989; Hogarth et al., 2013; Hogarth et al., 2011; McLean et al., 2002; Mitranond et al.,
1979; Unni et al., 1983; Van Pelt and De Rooij, 1990a, b). Conversely, precocious exposure
to RA at 1 or 2 dpp resulted in premature expression of stimulated by retinoic acid gene 8
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(STRA8) in prospermatogonia and a delay in meiotic entry when compared to vehicle

treated controls (Busada et al., 2014; Snyder et al., 2011) suggesting that prospermatogonia
may be vulnerable to extrinsic signals prior to the time of normal RA production. However,
the effects of aberrant testicular RA levels on the pool of SSCs in the neonatal mouse testis
have not been directly examined.

Here we show that reduced RA alters the number of SSCs present in the neonatal testis but
precocious RA exposure in the neonatal testis does not, and we present a model for how RA
regulates SSC establishment.

Materials and Methods

Animals
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Animal experiments were approved by the Washington State University Animal Care and
Use Committees and conducted in accordance with the guided principles for the care and
use of research animals of the National Institutes of Health. B6;129S- Gt(ROSA)26Sor/J
(mice designated ROSA; The Jackson Laboratory, Stock No: 002073), ID4-EGFP transgenic
reporter line (Chan et al., 2014), and C57BL/6-129ScvP mouse colonies were maintained in
a temperature- and humidity-controlled environment with food and water provided ad

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libitum. Animals were euthanized by CO2 asphyxiation followed by decapitation (0–10 dpp)
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or cervical dissociation (10–180 dpp), and their testes dissected.

WIN 18,446 and RA treatments

WIN 18,446 and RA treatments were performed as described (Hogarth et al., 2013; Snyder
et al., 2011). From 2 to 8 dpp, 100 μg/g body weight of WIN 18,446, suspended in 1% gum
tragacanth, was pipette fed daily to neonatal ID4-GFP or ROSA mice and euthanized at 9
dpp. Vehicle treated control animals received 1% gum tragacanth alone. Some WIN 18,446-
treated animals also received an intraperitoneal (IP) injection of 200 μg of RA, diluted in 10
μL of dimethyl sulfoxide (DMSO), to synchronize the onset of spermatogenesis, and were
left to recover for either 8 or 48 hours. Precocious RA exposure was induced in 2 dpp mice
by subcutaneous injection of 50 μg of RA, diluted in 10 μL of DMSO, and mice were
allowed 24 hours to recover prior to euthanasia. Vehicle control mice received a
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subcutaneous injection of 10 μL of DMSO.

Whole Mount Immunofluorescence

Whole mount immunofluorescence was performed as previously described (Agrimson et al.,
2016). ID4-EGFP testes were detunicated and the tubules were gently dissociated manually
with forceps under magnification using a dissecting microscope (Olympus Model SZX-
ILLD2-100, PA, USA). The dissociated tubules were fixed using 4% paraformaldehyde
(P6148, Sigma-Aldrich, MO, USA) with gentle rotation for 2 hours at 4°C and stored in IX
PBS (137mM NaCl/2.7mM KCl/10.1mM Na2HP04/1.8mM KH2PO4) at 4°C until use.
Primary antibodies were: goat anti-GFP-FITC (2.5 μg/mL, AB6662, Abcam, MA, USA) and
rabbit anti-STRA8 (1:2000 dilution, made in-house (Hogarth et al., 2013)). Secondary
antibody was Alexa Fluor reagent donkey anti-rabbit 488 (2 (μg/mL, A21206, Invitrogen,
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CA, USA), diluted in PB (1% bovine serum albumin/PBS). Confocal microscopy (LEICA
TCS SP5 II, IL, USA) was used to image whole seminiferous tubules and at least 3 neonatal
animals were examined to ensure consistent results. ID4-EGFP and STRA8-copositive
spermatogonia were quantified in testis cross sections following RA treatment. At least 250
round tubule cross sections within at least three sections, separated by 25 microns, from
three animals per treatment group were examined to determine the percentage of ID4-EGFP
+ prospermatogonia expressing STRA8. Paired Student T Tests (Microsoft Excel) were used
to determine significance between control and treated samples. A P-value less than 0.05 was
deemed significant.

Germ Cell Transplantation

Transplantation was performed as previously described (Helsel and Oatley, 2017).
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Recipients were C57BL6/JX129S1/svlmJ F1 hybrid mice treated with IP injection of

busulfan (50mg/kg of body weight) at 6 weeks of age and recovered at least 6 weeks to
ensure germ cell depletion (Brinster and Avarbock, 1994). Donors were Rosa26-LacZ
transgenics (obtained from Jackson laboratories, stock no. 002073), treated with either 7
consecutive daily treatments of WIN 18,446-only or a single RA-only injection at 2 dpp.
Single cell suspensions of testes were generated by enzymatic disassociation and the germ
cell population enriched by Percoll selection as described previously (Oatley and Brinster,
2006) and germ cells were re-suspended in mouse SSC serum-free medium (mSFM) at 106

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cells/mL. Approximately 7 to 10 μl of cell suspension was microinjected into seminiferous

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tubules via the rete testis. For each recipient, one testis received germ cells from WIN
18,446-only or RA-only donors and the contralateral testis received cells from a vehicle
control donor. Microinjection of treated or control cell suspensions was varied between the
right and left testis. Three transplantation studies were performed for each experimental
treatment and 2 to 6 recipient mice were used for each. Recipient mice were given at least 70
days to recover and allow for spermatogenic colony regeneration. Following euthanasia, the
recipient testes were detunicated, fixed with 4% paraformaldehyde for 2 hours at 4°C, and
washed and stained in bromo-chloro-indolyl-galactopyranoside (X-gal) as previously
described (McLean et al., 2002). Dark blue colonies of spermatogenesis were counted using
a dissecting microscope by gently separating the seminiferous tubules (Figure 2C). The
relative number of SSCs per donor testis was determined by multiplying the number of
colonies counted from each testis by the fold-difference in cells per mL isolated from treated
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testes versus control testes. Paired Student T Tests (Microsoft Excel) were performed to
determine significance between control and treated samples. A P-value less than 0.05 was
deemed significant. Transplantation analyses were performed at least 3 times from 3
different treated and control donor animals to achieve technical and biological replication.

Flow Cytometric Analysis

Single cell suspensions were prepared using trypsin/EDTA for testis digestion (Oatley and
Brinster, 2006) and re-suspended at a concentration of 1 × 106 cells/mL in DPBS-S. Treated
and control cell suspensions were then analyzed for EGFP intensity using a Cytoflex flow
cytometer (B53000, Beckman Coulter, CA, USA) and the CytExpert Software (Beckman
Coulter) to determine the percentage of ID4-EGFP+ and ID4-EGFP- cells. Additionally
within the ID4-EGFP+ cells, the percentage of ID4-EGFP Bright, Mid, and Dim cells
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between treatments was measured as described previously (Helsel et al., 2017). The analysis
was conducted at least 3 times from 3 different treated and control donor animals to achieve
technical and biological replication.

Results
RA deficiency alters the ratio of ID4-EGFP-bright and -dim spermatogonia and increases
SSC number in the neonatal testis
Vitamin A deficiency (VAD) and RA depletion by WIN 18,446 treatment both enrich testes
with undifferentiated spermatogonia (Evans et al., 2014; Griswold et al., 1989; Hogarth et
al., 2013; McLean et al., 2002; Mitranond et al., 1979; Unni et al., 1983; Van Pelt and De
Rooij, 1990a, b). However, the effect of RA deficiency on the ID4-EGFP expressing SSC
population has not been examined. We used whole mount immunofluorescence to
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qualitatively compare the ID4-EGFP populations in seminiferous tubules of control (RA

sufficient) and WIN 18,446-treated (RA deficient) mice. An abundance of apparently ID4-
EGFPBright SSCs were present in WIN 18,446 treated animals, while these cells were sparse
in vehicle treated controls (Figure 1A and 1B). After RA exposure of WIN 18,446 treated
animals 3 different populations of spermatogonia emerged within 8 hours: 1) ID4-
EGFPBright, 2) STRA8- and ID4-EGFPDim -co-positive, and 3) STRA8 only-positive
spermatogonia (Figure 1C). Forty-eight hours after RA the first and third cell populations

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were only present, consistent with differentiation of the STRA8- and ID4-EGFPDim -co-
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positive cells (Figure 1D).

To quantify the effects of RA deficiency on the ID4-EGFP+ population in the neonatal testis,
we used flow cytometric analysis to assess: 1) the overall percentage of EGFP+ cells in
testes from animals treated with WIN 18,446 compared to vehicle control, and 2) the
proportion of Bright, Mid, and Dim fluorescent cells within the ID4-EGFP+ population in
testes of animals from the two treatment conditions. The percentage of total ID4-EGFP+
cells was unchanged in the absence of RA (Figure 1E). However, the proportions of ID4-
EGFPBright and ID4-EGFPMid cells were significantly elevated and the proportion of ID4-
EGFPDim cells was significantly decreased in testes from WIN 18,446-only treated animals
compared to vehicle treated controls (Figure 1F). Collectively, these results indicate RA
deficient testes have increased numbers of ultimate SSCs and daughter cells in transition to a
progenitor state.
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To functionally test whether the increase in ID4-EGFPBright/Mid spermatogonia in RA

deficient animals reflects an increase in SSC number, we performed transplantation analysis.
We compared engraftment of germ cells from ROSA donor mice treated with either WIN
18,446 or vehicle (Figure 2A and 2B). Because each donor-derived colony in recipient testes
is clonally-derived from a single SSC, quantification of colony number provides a
retrospective measure of SSC content in the transplanted donor cell suspension. We found
that germ cells from both RA-deficient and RA-sufficient control testes could generate
spermatogenic colonies following transplantation (Figure 2A and 2B) and testes of RA
deficient mice contained a significantly greater number of SSCs compared to those of
controls (Figure 2D and 2E). Taken together, these results demonstrated an increased
number of true SSCs when RA is depleted.
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Precocious RA exposure induces STRA8 expression within a majority of the ID4-EGFP+

prospermatogonial population but does not alter the number of SSCs in neonatal testes
Next, we asked whether exogenous RA treatment can cause premature SSC establishment at
2 dpp. Using whole mount immunofluorescence, we observed that early exposure to RA
induced STRA8 expression in 80% of the ID4-EGFP+ prospermatogonia (Figure 3A and
3C), while only 45% of the ID4-EGFP+ spermatogonia in vehicle control mice expressed
STRA8 (Figure 3B and 3C). These results indicate that the ID4-EGFP+ population can
respond to RA by activating STRA8 expression, but do not address whether this reflects a
change in functional capacity. To assess this, we used flow cytometric analysis to quantify
ID4-EGFP expression. We found no significant difference in the percentage of germ cells
that were ID4-EGFP+ in testes exposed to excess RA compared to controls (Figure 3D) and
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no change in the abundance of Bright, Mid, or Dim cells in the presence of excess RA
exposure (Figure 3E).

Finally, we asked whether premature expression of STRA8 by ID4-EGFP+

prospermatogonia alters their capacity to regenerate spermatogenesis. We used
transplantation analyses to compare the SSC content in testes of mice exposed to excessive
RA versus controls. Donor mice at 2 dpp were treated with subcutaneous RA or vehicle
injection, germ cell suspensions were isolated 24 hr later, and a standard number of cells was

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transplantation into recipient testes. Both RA-treated and control donors generated colonies,
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demonstrating the presence of SSCs (Figure 4A and 4B) and calculation of engraftment
efficiency found no difference between RA-treated and control mice (Figure 4C and 4D).
Collectively, these data indicate that excessive RA in neonatal testes does not alter SSC
abundance.

Discussion
This study has, for the first time, provided evidence to suggest that RA availability may
influence SSC pool formation in the neonatal testis. During the onset of spermatogenesis,
germ cells with features of undifferentiated spermatogonia arise between 3 and 5 dpp
(Drumond et al., 2011; Kluin and de Rooij, 1981; Yoshida et al., 2006; Yoshida et al., 2004),
with the SSC pool eventually forming within this population. The exact timing of SSC pool
establishment is not known, but is proposed to occur between 0 and 6 dpp (Bellve et al.,
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1977; Chan et al., 2014; de Rooij and Russell, 2000; Huckins and Clermont, 1968; McLean
et al., 2003). RA is known to trigger spermatogonial differentiation and the onset of
spermatogenesis during this period, but whether RA regulates SSC pool establishment has
been unknown. Our results suggest that RA deficiency causes a greater proportion of
progenitor undifferentiated spermatogonia to retain their SSC state past the age when the
pool is thought to be determined.

While genetic models have been used to induce RA deficient testes in juvenile mice (Li et
al., 2011; Raverdeau et al., 2012), SSC abundance has not been examined in these models.
We recently showed that treatment with the aldehyde dehydrogenase enzyme inhibitor is a
highly effective method to eliminate RA production in neonatal testes (Hogarth et al., 2015;
Hogarth et al., 2013). Spermatogonial differentiation does not occur in mice treated with
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WIN 18,446 and consequently the testes become enriched with undifferentiated
spermatogonia (Hogarth et al., 2013). Work of Helsel et al., 2017 demonstrated through
functional transplantation analyses that the ID4-EGFPBright population is essentially pure
SSCs by 8 dpp. Here we found that ID4-EGFPBright spermatogonia are enriched in the testis
following WIN 18,446 treatment, suggesting an increased number of spermatogonia with
stem cell potential, a possibility we confirmed by transplantation. Taken together, flow
cytometry and transplantation analyses suggest that some ID4-EGFPMid spermatogonia still
retain regenerative capacity consistent with the proposal by Helsel et al., 2017 that ID4-
EGFP levels are proportional to SSC potential.

Based on our findings, we hypothesize that RA signaling in the neonatal testis promotes
formation of the SSC pool, either directly or indirectly. Previous work has shown that steady
state SSCs do not express RA receptor γ (RAR γ) and the earliest germ cell expression of
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RARγ occurs at 5 dpp (Gely-Pernot et al., 2012; Vernet et al., 2006), however expression of
the receptor has not been fully investigated in the prospermatogonial population between 1
and 5 dpp. To fully understand how RA availability may be fostering SSC pool formation
future studies will be necessary. Our data indicate that RA deficiency leads to more
undifferentiated spermatogonia retaining the potential to be SSCs. It is unlikely that this
increased regenerative potential is due to increased SSC proliferation, as we previously
found that the undifferentiated spermatogonial population exits the cell cycle upon WIN

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 Agrimson et al. Page 7

18,446 treatment (Agrimson et al., 2016). Transplantation studies have shown that the
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number of Sertoli cells in the testis correlates directly to the number of SSCs (Oatley et al.,
2011), and the Sertoli cells are known to synthesize the RA required for the onset of
spermatogenesis (Raverdeau et al., 2012). We suggest that RA from Sertoli cells may help,
at least initially, to coordinate Sertoli cell number with SSC pool size.

It is well known that excess RA exposure has a potent effect on spermatogenesis prior to the
time of preleptotene spermatocyte formation (Busada et al., 2014; Davis et al., 2013; Snyder
et al., 2011), including early expression of STRA8 in prospermatogonia and delayed
progression of spermatogenesis (Busada et al., 2014; Snyder et al., 2011). It has also been
suggested that the delay is because excess RA induces RA degrading enzymes, thus slowing
differentiation (Busada et al., 2014). We found that 24 hours after RA treatment, ID4-EGFP
expressing prospermatogonia also expressed STRA8, suggesting that this population of
germ cells are susceptible to the RA trigger in terms of induction of a hallmark gene
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response. However, we observed no difference in the proportion of ID4-EGFPBright

prospermatogonia or the number of regenerative spermatogonia as defined by
transplantation. Moreover, excess RA did not shift the populations of ID4-
EGFPBright/Mid/Dim prospermatogonia. Thus, RA induction of STRA8 gene expression does
not necessarily indicate a functional change in SSC potential.

We found that RA depletion affects SSC abundance in neonates but RA supplementation

does not. To explain these results, we suggest that at least three populations of
spermatogonia derive from prospermatogonia: differentiating spermatogonia destined to
form the first wave of spermatogenesis, SSCs, and progenitor undifferentiated
spermatogonia (Figure 2F and 4E). We suggest that the progenitor cells are responsive to RA
depletion and retain the capacity to form SSCs (Figure 2G). In contrast, prospermatogonial
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exposure to excess RA causes a reversible induction of STRA8, via an unknown mechanism

that prevents differentiation of the ultimate SSCs (Figure 4F). This response likely
coordinates with the delay in timing of meiotic entry (Busada et al., 2014; Snyder et al.,
2011). Recent work has shown that within the RA deficient testes neither SSC niche
availability nor excessive germ cell proliferation can explain the increased SSC number
following WIN 18,446-only treatment (Agrimson et al., 2016). Ultimately, understanding
the dynamics of the ID4-EGFP+ population will help reveal how RA availability affects the
establishment of the SSC pool.

Acknowledgments
The authors would like to thank Amy Kaucher and Aileen Helsel for their assistance during the germ cell
preparations for SSC transplantations, Dr. Qi-En Yang for his technical assistance regarding counting SSC colonies
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following transplantation, and Dr. David Zarkower for critically reading the manuscript.

Funding: This work was supported by the National Institutes of Health [Grants R01 HD10808 to MDG and R01
HD061665 to JMO.

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Figure 1. RA deficiency alters the dynamics of the ID4-EGFP+ population in the mouse neonatal
testis
Representative images of whole mount testis tubules from mice treated with WIN 18,446-
only (A), vehicle control (B), WIN 18,446 followed RA for 8 hours (C) or 48 hours (D). All
tubules were stained for both STRA8 and EGFP. Blue arrows denote what appear to be ID4-
EGFPBright+/STRA8- germ cells, yellow arrows indicate ID4-EGFPDim+/STRA8+ germ
cells, and purple arrows designate ID4-EGFP-/STRA8+ spermatogonia. n=3 different mice
for each treatment. Bars = 50 μm. (E) Quantitative comparison from flow cytometric
analysis of the percentage of total ID4-EGFP+ and ID4-EGFP- cells in testes of mice treated
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with WIN 18,446-only and vehicle control. (F) Quantitative comparison of the percentage of
the ID4-EGFP+ population that could be classified as Bright, Mid, or Dim in mice treated
with WIN 18,446-only or vehicle control. Data are mean±SEM for three independent
experiments. * denotes significantly different at a p-value < 0.05.

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Figure 2. Alteration of SSC content in the RA deficient neonatal mouse testis

Representative images of recipient testes 70 days following transplantation that have been
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stained for colonies of donor-derived spermatogenesis from germ cells isolated after
treatment with (A) WIN 18,446-only or (B) vehicle control. (C) Image depicts how dark
blue colonies of spermatogenesis, shown by the brackets and black arrows, were counted
within each recipient testis. (D) Box and whisker plot shows the quantitation of the number
of donor-derived colonies from germ cells collected from either WIN 18,446-only treated or
vehicle control treated mice. (E) Quantitative comparison of number of SSCs in testes of
mice treatment with WIN 18,446-only or the vehicle control. Data are mean±SEM and n=3

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different cell preparations and 10 recipient mice (each individual recipient mouse is labeled
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differently as white, black or red squares, triangles, circles, or diamonds). * denotes

significantly different at a p < 0.05. (F) Hypothesized model depicting the derivation of 3
distinct populations (SSCs, progenitors, and differentiating spermatogonia) from
prospermatogonia in the neonatal testis. Black oval indicates prospermatogonia. Dark,
medium, and light green ovals depict ID4-EGFP bright (SSCs), mid (cells transitioning from
SSC to progenitor), and dim (progenitor) cells, respectively. Red ovals mark STRA8-
positive differentiating spermatogonia. (G) Hypothesized model depicting the effects RA
deficiency on ID4-EGFP + germ cell dynamics during SSC pool establishment. Black oval
indicates prospermatogonia. Dark, medium, and light green ovals depict ID4-EGFP bright
(SSCs), mid (cells transitioning from SSC to progenitor), and dim (progenitor) cells,
respectively.
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Figure 3. Induction of STRA8 expression in ID4-EGFP+ prospermatogonia following exposure

to excess RA does not alter the dynamics of the ID4-EGFP+ germ cell population in the neonatal
testis
Representative images of whole mount testis tubules from mice treated with exogenous RA
(A) or vehicle control (B) and stained by immunofluorescence for STRA8 (red) and ID4-
EGFP (green). Blue arrows denote ID4-EGFP+/STRA8- cells and yellow arrows indicate
ID4-EGFP+/STRA8+ cells. Bar = 50 μm. (C) Quantitative comparison of the percentage of
ID4-EGFP only-positive prospermatogonia. (D) Quantitative comparison from flow
cytometric analysis of the percentage of total ID4-EGFP+ and ID4-EGFP- cells in testes of
mice treated with vehicle or exogenous RA. (E) Quantitative comparison of the percentage
of the ID4-EGFP+ population that can be classified as Bright, Mid, or Dim in testes of mice
treated with vehicle or exogenous RA. Data are mean±SEM and n=3 different mice for each
treatment. *denotes significantly different at a p = 0.005.
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Figure 4. Excess RA exposure in the neonatal mouse testis does not alter SSC number
Representative images of recipient testes 70 days post-transplantation that have been stained
for colonies of donor-derived spermatogenesis from germ cells isolated after treatment with
(A) RA or (B) vehicle control. (C) Quantitation of the number of donor-derived colonies
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generated by germ cell suspensions from testes of mice treated with exogenous RA or
vehicle control. (D) Quantitative comparison of the number of SSCs present in testes of mice
treated with exogenous RA or vehicle control. Data are mean±SEM and n=3 different cell
preparations and 14 recipient mice (each individual recipient mouse is labeled differently as
white, black, blue or red squares, triangles, circles, or diamonds). (E) Hypothesized model
depicting the derivation of 3 distinct populations (SSCs, progenitors, and differentiating
spermatogonia) from prospermatogonia in the neonatal testis. Black oval indicates

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 Agrimson et al. Page 15

prospermatogonia. Dark, medium, and light green ovals depict ID4-EGFP bright (SSCs),
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mid (cells transitioning from SSC to progenitor), and dim (progenitor) cells, respectively.
Red ovals mark STRA8-positive differentiating spermatogonia. (F) Hypothesized model
depicting the effects of excess RA exposure on ID4-EGFP + germ cell dynamics during SSC
pool establishment. Black oval indicates prospermatogonia. Dark, medium, and light green
ovals depict ID4-EGFP bright (SSCs), mid (cells transitioning from SSC to progenitor), and
dim (progenitor) cells, respectively. Dark and light yellow ovals show ID4-EGFP and
STRA8 co-positive cells. Red ovals mark STRA8-positive differentiating spermatogonia.
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