TOXICOLOGICAL SCIENCES 117(1), 218–224 (2010)
Advance Access publication June 18, 2010
Human Primordial Germ Cell Formation Is Diminished by Exposure to
Environmental Toxicants Acting through the AHR Signaling Pathway
Kehkooi Kee,*,1,2,3Martha Flores,*,3Marcelle I. Cedars,† and Renee A. Reijo Pera*
*Department of Obstetrics and Gynecology, Center for Human Embryonic Stem Cell Research and Education; Institute for Stem Cell Biology and Regenerative
Medicine, Stanford University School of Medicine, Stanford University, Palo Alto, California; and †Department of Obstetrics, Gynecology and Reproductive
Sciences, Center for Reproductive Health, University of California, San Francisco, California 94115
1Present address: School of Medicine, Tsinghua University, Beijing 100084, China.
2To whom correspondence should be addressed. Fax: 10-627-94200. Email: email@example.com.
3These authors contributed equally.
Received May 20, 2010; accepted June 7, 2010
Historically, effects of environmental toxicants on human
development have been deduced via epidemiological studies
because direct experimental analysis has not been possible.
However, in recent years, the derivation of human pluripotent
stem cells has provided a potential experimental system to directly
probe human development. Here, we used human embryonic stem
cells (hESCs) to study the effect of environmental toxicants on
human germ cell development, with a focus on differentiation of
the founding population of primordial germ cells (PGCs), which
will go on to form the oocytes of the adult. We demonstrate that
human PGC numbers are specifically reduced by exposure to
polycyclic aromatic hydrocarbons (PAHs), a group of toxicants
common in air pollutants released from gasoline combustion or
tobacco smoke. Further, we demonstrate that the adverse effects of
PAH exposure are mediated through the aromatic hydrocarbon
receptor (AHR) and BAX pathway. This study demonstrates the
utility of hESCs as a model system for direct examination of the
molecular and genetic pathways of environmental toxicants on
human germ cell development.
Key Words: human embryonic stem cells; germ cell
differentiation; reproductive toxicology; polycyclic aromatic
hydrocarbons; aromatic hydrocarbon receptor (AHR).
Polycyclic aromatic hydrocarbons (PAHs) are one of the
most common components of air pollution and are formed
during the incomplete burning of organic substances (coal, oil,
tobacco, and meat); they enter the environment mainly as
exhaust from automobiles, residential wood burning, forest
fires, or secondhand cigarette smoke (Finlayson-Pitts 1997).
Numerous studies have linked PAH exposure to tumorigenic-
ity, reproductive failure, and developmental birth defects in
laboratory animals (Castro et al., 2008; Detmar et al., 2008;
Jacobsen et al., 2008). In parallel, epidemiological studies have
linked human PAH exposure to lowered reproductive capacity,
pulmonary disease, tumorigenicity, birth defects, and behav-
ioral abnormalities (Fowler et al., 2008; Millman et al., 2008;
Perera et al., 2005a,b, 2007). However, although animal and
epidemiological studies indicate adverse outcomes in associ-
ation with PAH exposure, tools to directly assay adverse
outcomes of PAH exposure during development of particular
human cell lineages have not been available.
A series of studies in mice have demonstrated that oocytes
and fetal germ cells in mice are susceptible to exposure to
PAHs (Jurisicova et al., 2007; Matikainen et al., 2001, 2002;
Pru et al., 2009). Moreover, studies indicate that PAH-
mediated oocyte destruction could be prevented by inactivation
of the apoptotic gene, Bax, and depended on the aromatic
hydrocarbon receptor (AHR) to activate Bax expression
(Matikainen et al., 2001, 2002). More recently, mouse fetal
germ cells were also shown to apoptose in response to
incubation with PAHs; however, fetal germ cell toxicity could
be prevented by the selective AHR antagonist, a-napthoflavone
(ANF) (Coutts et al., 2007; Jurisicova et al., 2007; Matikainen
et al., 2001, 2002). Taken together, these studies indicate that
PAH exposure can severely reduce the number of developing
fetal germ cells and oocytes in mammals (Coutts et al., 2007;
Matikainen et al., 2001, 2002). Here, we addressed whether
differentiation of human embryonic stem cells (hESCs) to the
germ cell lineage, which ultimately gives rise to mature eggs
and/or sperm, is altered in the presence of PAHs and/or PAH
MATERIALS AND METHODS
hESC differentiation and treatment. hESC line (H9, XX) was maintained
and differentiated as previously described (Kee et al., 2006, 2009). Briefly,
adherent differentiation began upon the addition of differentiation media
containing 20% fetal bovine serum (Invitrogen, Inc., Carlsbad, CA) and
supplemented with Bone Morphogenetic Proteins (BMPs) 4, 7, and 8b (R&D
Systems, Minneapolis, MN), reconstituted in 4mM HCl/0.1% bovine serum
albumin, and used at 50 ng/ml. 9,10-Dimethylbenz[a]anthracene (DMBA;
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Sigma, Inc., St Louis, MO), ANF (Sigma), and DMBA-3,4-dihydrodiol
(DMBA-DHD; Midwest Research Institute, Kansas City, MO) were dissolved
in dimethyl sulfoxide (DMSO) before adding to the media at 1:1000 dilution
with indicated final concentrations.
High-density Real Time-PCR/Quantitative PCR analysis by Fluidigm.
Total RNA was extracted using the RNeasy kit (Qiagen, Inc., Valencia, CA)
and complementary DNA (cDNA) prepared with SuperScriptIII (Invitrogen)
according to the manufacturer’s protocols using 1 lg RNA. The cDNA was
subjected to a preamplification using 1.25 ll out of 20 ll total cDNA, 1 ll
Platinum Taq (Invitrogen), 5 ll CellsDirect 23 Reaction Buffer, and 2.5 ll
0.23 Taqman (Applied Biosystems, Foster City, CA) probe mix. PCR cycle
program for preamplification is as follows: 95?C, 10 min; 95?C, 15 s; and 60?C,
4 min for 14 cycles. Assays and samples are prepared according to the Fluidigm
protocol and run on a 48 3 48 chip. BioMark (Fluidigm, South San Francisco,
CA) program was used to obtain delta Ctvalue before imported into Excel file
sheet to calculate delta-delta Ctvalue ¼ 2ðCtGene?CtHousekeepinggenesÞ. The delta-delta
Ctvalue is then normalized to the control of each experiment to obtain the final
normalized expression level. All delta-delta Ctvalues were calculated using
four housekeeping genes (GAPDH, CTNNB1, ACTB, and CENTRIN) in high-
density RT-PCR/quantitative PCR (qPCR) analysis using the microfluidic
Fluidigm system. In contrast, the experiment in Figure 2 only used GAPDH as
a housekeeping gene. The use of GAPDH alone was employed in this case as
Figure 2 demonstrates results of standard qPCR reactions in a 7300 Real-Time
PCR System (Applied Biosystems) used for screening of short hairpin RNAs
(shRNAs) for further analysis.
Western analysis of human AHR. Cells were collected in prechilled PBS
with Complete Mini Protease Inhibitor (Roche Applied Science, Inc., Indian-
apolis, IN) followed by centrifugation for 3 min at 5000 rpm in microcentrifuge at
4?C. Supernatant was removed and pellet resuspended in 200 ll RIPA buffer and
stored at ?80?C. Samples were thawed and centrifuged again before the
supernatant was subjected to bicinchoninic acid protein concentration measure-
ment (Pierce Biotechnology, Inc., Rockford, IL). Thirty-five micrograms of
protein was loaded on an 8% SDS-polyacrylamide gel electrophoresis
and transferred onto polyvinylidene fluoride membrane for 1 h at 100 V in
N-cyclohexyl-3-aminopropane-sulfonic acid (CAPS) buffer (10mM CAPS, 20%
methanol, pH 11). The membrane was blocked overnight in 5% nonfat milk at
4?C. Mouse monoclonal antibody to AHR (Abcam, Cambridge, MA) was diluted
to 1:1000 in 5% nonfat milk followed by goat anti-mouse secondary horse radish
peroxidase (Zymed [Invitrogen], Carlsbad, CA) at 1:20,000. Illuminated signal
was detected using the ECL Plus System (Amersham, Piscataway, NJ).
shRNA vectors and preparation of lentivirus. shRNA was used to target
AHR by the BLOCK-iT Inducible H1 Lentiviral RNAi System (Invitrogen).
Double-stranded oligos were generated, ligated into the pENTR vector, and
transfected into 293FT cells for initial screening. After 24 h, RNA was
harvested using the RNeasy kit and cDNA generated using SuperScriptIII with
1 lg total RNA input. The destination lentiviral vector was generated by
recombining the pENTR vector with the pLenti4/BLOCK-it-DEST vector via
the Gateway technology according to the manufacturer’s protocol. Lentiviral
supernatant carrying the pLenti4/BLOCK-it-DEST-shAHR vector was gener-
ated by cotransfection with 10 lg of each vector with 10 lg Vsvg and 15 lg
D8.9 into 293FT cells grown on T175. Supernatant was harvested after 3 days
and ready for transduction into hESCs or frozen at ?80?C until further usage.
hESCs prepared for transduction were plated to 50% confluency on matrigel-
coated plates. Polybrene was added to the lentivirus supernatant for a final
concentration of 8 lg/ml. A 0.5 ml of the mixture was incubated with hESCs in
a well of six-well plate for 6 h at 37?C before adding 2.5 ml conditioned media
(hESCs media incubated overnight with irradiated MEFs). hESCs were
incubated overnight before being washed 23 with PBS and replacing with new
conditioned media. The next day, Zeocin was added at 2 lg/ml final concen-
tration to new conditioned media, and the transduced hESCs were selected for
3 days before beginning differentiation as described above.
Fluorescence-activated cell sorting analysis and Caspase 3/7 assay.
Single-cell suspensions were prepared first by incubating differentiated hESCs
in Collagenase Type IV (1 mg/ml) for 10 min followed by 10 min TrypLE
(Invitrogen) treatment. Cell pellet was resuspended in 0.5 ml differentiated
media and passed through a 40-lm strainer. Cell suspensions were then
subjected to fluorescence-activated cell sorting (FACS) analysis with BD
FACSAria system (BD Biosciences, San Jose, CA). Cells were sorted for either
VASA:GFPþ or VASA:GFP?. One thousand cells of each group were
collected in 100 ll PBS and mixed with 100 ll of Caspase-Glo 3/7 reagent
according to manufacturer’s protocol (Promega, Madison, WI). Luminescence
was measured after 1 h of incubation at room temperature with Fluostar Optima
(BMG, Offenburg, Germany).
To examine the effect of PAH exposure on human germ cell
development, we tested whether the prototypical PAH, DMBA,
or its metabolite, DMBA-DHD, affected germ cell differen-
tiation from hESCs. Our previous studies indicated that
expression of human germ cell–specific genes, including VASA
and DAZL, is induced by culture with BMP-4, -7, and -8b (Kee
et al., 2006). We observed that addition of either DMBA or
DMBA-DHD at the concentrations used previously in mouse
studies (Matikainen et al., 2001) reduced the expression of the
early germ cell–specific genes, VASA, DAZL, and PRDM1
(BLIMP1) (Fig. 1). Expression of VASA and DAZL decreased
to ~0.2 to 0.02 of control levels, respectively, with a lesser
effect observed for PRDM1 (0.4–0.06 of control). Consistent
with previous studies in mice in which DMBA-DHD was more
potent than DMBA (Matikainen et al., 2001), in our studies, we
observed a similar decrease in germ cell gene expression with
1lM DMBA and 0.1lM DMBA-DHD and observed that 1lM
DMBA-DHD was more potent than 10lM DMBA. This
indicated that DMBA-DHD is at least one log more potent than
DMBA, as measured by the decrease in germ cell–specific
gene expression (Fig. 1). Concurrent with analysis of germ
cell–specific gene expression, we also analyzed expression of
the apoptotic gene, BAX, and two somatic cell markers, NES
and KDR. We observed that expression of BAX increased 3- to
16-fold with addition of DMBA and DMBA-DHD, respec-
tively. When the antagonist of AHR, ANF, was added with
DMBA-DHD, BAX expression decreased, whereas that of the
germ cell lineage markers VASA, DAZL, and PRDM1 increased
relative to DMBA-DHD addition alone. Although the rescue
by ANF did not restore germ cell expression to the same level as
controls, the partial rescue strongly suggested that DMBA-DHD
acted through the AHR pathway. In contrast, we noted that both
DMBA and DMBA-DHD did not significantly alter expression
of the two somatic gene markers KDR (mesodermal marker) and
NES (ectodermal marker), and moreover, no rescue by ANF was
observed. Taken together, these observations indicate that PAH
addition to differentiating hESCs resulted in a specific decrease
in expression of germ cell–specific genes that are diagnostic of
primordial germ cells (PGCs).
To test if the decreased germ cell gene expression was
mediated through the AHR signaling pathway, we constructed
short hairpin RNAs to silence AHR expression. By disrupting
PAH EXPOSURE AND HUMAN GERM CELL DEVELOPMENT
the essential component of the apoptosis pathway, we expected
that the adverse affect of PAH addition to differentiating
human germ cell cultures would be alleviated. Five shRNA
sequences were chosen to target different regions of human
AHR (Fig. 2A), subcloned and tested in 293FT cells for their
silencing effect on AHR. qPCR measurement of AHR transcript
levels indicated significant reduction by all short hairpin AHRs
(shAHRs) (Fig. 2B), with reductions to approximately 0.8- to
0.3-fold of the control (a silencing vector carrying LacZ
targeting sequence). shAHR25 showed the greatest silencing
effect followed by shAHR16, shAHR7, shAHR15, and lastly,
shAHR10. We recombined shAHR15, 16, and 25 separately
into our destination vectors and examined silencing further by
Western analysis in both 293FT cells and hESCs (Fig. 3). We
noted that Western analysis with lysates of 293FT resulted in
detection of two prominent bands that migrated to the expected
size of AHR, ~95 kDa, whereas only one protein band was
detected in the lysates of hESCs. We observed that a reduction
in both the upper and lower bands occurred in 293FT cells and
were similarly reduced by all three shAHRs, suggesting that
both bands represent legitimate isoforms of AHR, likely to be
differentially posttranslationally modified in AHR in 293FT
cells. More importantly, shAHR16 and shAHR25 both reduced
AHR protein levels in hESCs more than shAHR15 and the
control, shLacZ, consistent with the qPCR analysis of AHR
transcript in 293FT cells.
We then proceeded to examine the effect of silencing AHR
on human germ cell differentiation from hESCs in the presence
of DMBA-DHD (Fig. 4) and observed that expression of AHR
was reduced to approximately 50% when shAHR25 was
transduced into hESCs and cells were differentiated for 14 days
with BMPs. VASA and DAZL expressions were significantly
elevated (rescued) when AHR was silenced in the presence of
DMBA-DHD. Expression of PRDM1 was also rescued by
shAHR, but to a lesser extent.
To further examine the effects of DMBA-DHD and shAHR at
the cellular level in terms of germ cell numbers, we used
a VASA:GFP reporter system to harvest human PGCs and
quantify differentiation (Kee et al., 2009). For this purpose,
hESCs were transduced with the VASA:GFP reporter and
selected for stable integration. Silencing vectors against AHR or
the control, LacZ, were introduced into the hESCs, and the cells
were differentiated in the presence or absence of DMBA-DHD.
In FACS, the majority of differentiated control hESCs reside on
the diagonal axis of the FACS plots when the phycoerythrin and
fluorescein isothiocyanate (FITC) channels were used to isolate
in differentiated hESCs induced by BMPs to germ cells. Controls are differentiated cells treated only with the solvent, DMSO. DMBA, DMBA-DHD, and ANF are
dissolved in DMSO before addition to the differentiating cells. Gene expression is measured by qPCR and normalized first to four housekeeping genes (GAPDH,
CTNNB1, ACTB, and CENTRIN) followed by normalization to the controls. Error bars are standard deviations from triplicates. Asterisk indicates averages from
three independent samples, significantly different from respective controls, by one-way ANOVA analysis; p < 0.05.
Expression of early germ cell genes is reduced in the presence of PAHs. Normalized gene expression of VASA, DAZL, PRDM1, BAX, NES, and KDR
KEE ET AL.
VASA:GFP cells (Fig. 5). In hESCs carrying the VASA:GFP
reporter, a population of cells also resided on the FITC side of
the plot when differentiated and was designated as the
VASA:GFPþ cells. Extensive characterization has demonstrated
that this population has properties of PGCs, including diagnostic
gene expression, methylation status at the imprinted loci and
genome-wide, alkaline phosphatase activity, and ability to form
embryonic germ cell lines (Kee et al., 2009). We observed
that approximately 4.5% of cells were VASA:GFPþ PGCs after
14 days of BMP induction in cells carrying the control vector,
shLacZ, without DMBA-DHD treatment (Fig. 5). When hESC
cultures were treated with DMBA-DHD, the percentage of
VASA:GFPþ PGCs was reduced to 3.1% (the difference in
terms of real numbers of PGCs was approximately 908 PGCs
out of 20,000 differentiated hESCs in the control vs. 626 PGCs
in the DMBA-DHD–treated group). On the other hand, cells
carrying shAHR25 showed 7.6 and 7.1% of VASA:GFPþ cells
with or without addition of DMBA-DHD. Thus, shAHR not
only rescued the reduction of VASA:GFPþ population but also
elevated the level of VASA:GFPþ compared with the control
cells. These results mirror those that reported an elevated
number of primordial oocytes in Ahr knockout mice (Robles
et al., 2000), again highlighting the AHR pathway as the major
apoptotic pathway of mammalian germ cells. As further
illustrated, a direct comparison of the number of VASA:GFPþ
cells in all four treatment groups demonstrates a significant
reduction of VASA:GFPþ at the same GFPþ intensity in the
DMBA-DHD–treated shLacZ group but not in the shAHR
groups (Fig. 5).
Finally, we sought to determine whether the adverse effect of
PAH exposure was specific to human germ cell differentiation
by further analysis of apoptosis in the isolated VASA:GFPþ
PGC population versus the VASA:GFP? population (Fig. 6).
We observed that apoptosis activity, measured by quantitative
AHR expressions in 293FT cells with control silencing vector, shLacZ, and five shAHR targeting different regions of AHR exons. Two amounts of shAHR, 0.5 and
1 lg, were transfected into 293FT, and qPCR of AHR gene expressions were measured after 24 h. One-microgram shLacZ transfection was used as control and for
normalization. Asterisk indicates averages from three independent samples, significantly different from respective controls by one-way ANOVA analysis; p < 0.05.
AHR is silenced in 293FT cells and hESCs. (A) Location of shRNA targeting sequences on the messenger RNA transcript of AHR. (B) Normalized
blots of GAPDH as loading controls of the same samples. Asterisk indicates second band, which may be a posttranslationally modified form of AHR in 293FT
Western analysis of AHR in 293FT and hESCs. Upper panels are Western blots against AHR in 293FT cells and hESCs, and lower panels are Western
PAH EXPOSURE AND HUMAN GERM CELL DEVELOPMENT
Caspase 3/7activity, was significantly increased by the addition
of DMBA-DHD in the VASA:GFPþ cells but not in the
VASA:GFP? cells. Moreover, this increase of apoptotic
activity was reduced by silencing of AHR with shAHR16 and
shAHR25, confirming that the pathway acted through AHR
and was specifically altered in the PGC population.
Results described here demonstrate that exposure to PAHs,
adversely and significantly, affects human PGC differentiation
from hESCs. These results also clearly demonstrate that the
molecular mechanism underlying reduction in both germ cell–
specific gene expression and germ cell numbers is linked to
AHR and apoptosis of PGCs. Moreover, we noted that
increased apoptosis induced by PAHs was specific to PGCs
and was not detected in the somatic population. Thus, our
analysis demonstrated that the clear detrimental effect of PAHs
on hESC differentiation is lineage specific. We note, however,
that addition of either DMBA or DMBA-DHD at the
concentrations used was based on previous studies in the
mouse (Matikainen et al., 2001); data regarding human fetal
exposures are lacking.
Although the specific decrease of gene expression of germ
cell markers was drastic and the apoptosis activity in PGCs
(VASA:GFPþ cells) was significantly higher than somatic
cells (GFP? cells), the reduction in human PGCs seemed
differentiated hESCs treated with 0.1lM DMBA-DHD carrying either control
silencing vector shLacZ or shAHR25. Asterisk indicates average from three
independent samples, significantly different from respective controls by one-
way ANOVA analysis; p < 0.05.
Gene expressions of AHR, DAZL, VASA, and PRDM1 in
positive cells are indicated on each plot of 20,000 cells, with the number of VASA:GFPþ in parenthesis. Negative control is hESCs without VASA:GFP reporter.
shLacZ or shAHR25 was transduced into hESCs with or without 1lM DMBA-DHD. (b) Histogram plot of the VASA:GFP–positive populations from (a)
comparing intensities and number of cells in 20,000 sorted cells.
(a) Silencing of AHR specifically rescues VASA:GFP cells. FACS plots of differentiated hESCs carrying VASA:GFP reporter. Percentages of GFP-
KEE ET AL.
minor in the presence of DMBA-DHD. This may be explained
if the remaining population of GFPþ cells (3.1% total) includes
the population of cells undergoing apoptosis but still intact and
viable. In that case, the adverse effect on VASA:GFPþ cell
imposed by PAHs could be more severe than the apparent
decrease by percentage of VASA:GFPþ cells. We also contrast
our results with follicular atresia. Follicular atresia is a process
that appears to be regulated in large part via hormonal apop-
totic pathways/processes acting through other receptor path-
ways, such as the tumor necrosis factor-alpha ligand/receptors,
Fas ligand/receptors, and others (Kaipia and Hsueh 1997). In
contrast, AHR-mediated apoptosis is a chemical-induced apop-
tosis with ligands of aromatic hydrocarbons and acting through
a distinct signaling pathway.
Even though recent studies suggested that pluripotent stem
cells might replenish depleted oocyte populations endowed at
birth (Johnson et al., 2004, 2005), independent analysis indicated
that replenishment of the mammalian germ cell population in
females does not occur under normal physiological conditions
(Eggan et al., 2006). Hence, the consequences of reducing germ
cell numbers by 30–50% in humans in vivo would be expected to
strongly and negatively impact fertility because of the limited
number of available oocytes at birth and the subsequent decline
in numbers until menopause in women. Indeed, in recent studies,
we demonstrated that silencing of the germ cell–specific gene,
DAZL, results in a twofold reduction in germ cell numbers
associated with aberrant expression of germ cell genes in vitro
(Kee et al., 2009). This genetic analysis, in vitro, supports results
of population-based studies on reproductive status and DAZL
polymorphisms (Tung et al., 2006a,b). Thus, it appears that
differentiation of hESCs to the germ line can provide an
experimental system for both genetic analysis of germ cell
formation as previously shown (Kee et al., 2009) as well as
analysis of environmental toxicology as shown here.
Notably in this study, we observed that by silencing AHR, we
could directly examine the effect of environmental perturbation
and molecular pathway of the adverse effect on germ cell
formation, which has never been feasible before on a human
genome background. Although gene expression was reduced to
50% (not as complete as the previously reported mouse
knockout; Robles et al., 2000), we observed a similar re-
quirement for AHR in hESC differentiation as observed in
mouse knockout models. Given the extensive epidemiological
data that have been collected retrospectively, in most cases,
indicating adverse fetal effects of maternal exposure to PAHs,
methods to directly assess lineage-specific human developmen-
tal defects are much needed. These studies suggest that at least
in regards to PAH exposure, the hESC system allows robust
examination of environmental factors or extrinsic factors on
human development, especially the germ cell lineage in early
development. This opens the door for large-scale toxicological
or drug-screening studies for basic or clinical reproductive
biology. In the future, investigation of applications to other types
of pluripotent stem cells such as induced pluripotent stem cells
and analysis of response of individual genetic composition to
environmental perturbation is also merited.
California Tobacco Related Disease Research Program
(TRDRP) postdoctoral fellowship to K.K; National Institutes
of Health National Institute for Child Health and Human
R.A.R.P.); TRDRP (14RT-0159 to R.A.R.P.); California
Institute for Regenerative Medicine (RC1-00137 to R.A.R.P.).
We are grateful to Dr Richard Anderson for providing
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