Diethylstilbestrol alters the population dynamic of neural precursor cells in the neonatal male rat dentate gyrus.
ABSTRACT Little is known about how estrogens influence neurogenesis in the newborn male rodent. Herein, we examined the effects of neonatal diethylstilbestrol (DES) exposure on the proliferation and survival of type-1 and type-2 neural precursor cells (NPC) in the dentate gyrus of male rats. This was achieved by exposing newborn male pups to DES on postnatal day (PND) 1, PND3, PND5, and PND7, sacrificed at PND8 or PND21, followed by double immunohistochemistry and morphometric analysis of hippocampal dentate gyrus. Furthermore, vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) mRNA expression was evaluated in hippocampal tissue blocks by real time RT-PCR. At PND8, the density of total proliferating NPC decreased in DES-treated animals. This reduction was due to a significant decrease in the mitotic rate of type-2 cells only, since type-1 NPCs did not show changes in the proliferation index. Type-2 NPCs expressed the cell-cycle inhibitor p27(kip1) and its expression was clearly augmented in the DES-treated group. Furthermore, the number of apoptotic cells in the dentate gyrus of DES-treated rats decreased. Surprisingly, DES treatment enhanced cell survival and increased NPCs proliferation when animals were examined 14 days after treatment. VEGF mRNA expression showed a positive correlation with NPCs proliferation and BDNF mRNA levels were higher in DES-treated animals at both time points examined. Collectively, these results indicate that hippocampal NPCs proliferation and survival is a critical target of DES exposure during the early postnatal period. VEGF and BDNF are proposed as key mediators of DES-induced NPC mitotic response.
- SourceAvailable from: Enrique H Luque[Show abstract] [Hide abstract]
ABSTRACT: Estradiol (E) has been suggested to have a neuroprotective effect in young animals but has neutral or harmful effects when it is administered to aged animals. In the present study, we determined whether the post-ovariectomy (post-OVX) timeframe elapsed before the initiation of chronic E treatment is critical for the estrogenic induction of neurotrophins (brain-derived neurotrophic factor, BDNF, and synaptophysin, SYN) in the rodent hippocampus. Adult mice were OVX and, a short period (short-term E (STE) animals) or a long period (long-term E (LTE) animals) after the OVX, were daily treated with E. Control animals were treated with sesame oil (short-term control (STC) and long-term control (LTC) animals). Protein expression was determined using an immunohistochemical approach. Transcriptional activity in the hippocampus of individual BDNF promoters was assessed by real-time quantitative RT-PCR, and the methylation levels of regulatory regions were analyzed by methylation-specific PCR and combined bisulfite restriction analysis. STE animals showed increased BDNF and SYN protein expression and a higher activity of BDNF II, IV and V promoters. In contrast, LTE animals did not show E induction of neurotrophins. In these animals, the methylation levels of regulatory sequences of the BDNF were higher than in the STE animals in a CpG island of promoter V and in the CRE regulatory site located in promoter IV. With this experiment, we determined that a prolonged period of hypoestrogenicity disrupts the E-induction of neurotrophins, and we postulated that DNA methylation is one of the epigenetic mechanisms that could explain the E-insensitivity of the BDNF after a long period post-OVX.The Journal of steroid biochemistry and molecular biology. 08/2014;
- [Show abstract] [Hide abstract]
ABSTRACT: Diethylstilbestrol is an estrogenic endocrine disrupter that has diverse health effects in humans. Bisphenol A is another estrogen-like chemical with possible similar effects to diethylstilbestrol, which has been increasingly used for industry to lead to globally widespread human exposure to it. Hematopoiesis is another of their possible targets, since estrogen suppresses erythropoietin induction to induce anemia. The aim of this study was to clarify the effects of diethylstilbestrol and bisphenol A on erythropoietin induction in rats. We observed the effects of one-shot subcutaneous injection of diethylstilbestrol or bisphenol A on hypoxia-, bleeding-, and cobalt-stimulated erythropoietin induction within 24 h and the hematological outcomes after repeated subcutaneous injection of diethylstilbestrol three times a week for 1 month in rats. Diethylstilbestrol at 10-1,000 μg/kg suppressed stimulus-elevated levels of plasma erythropoietin and its renal mRNA induction. In contrast, bisphenol A at 1,000 μg/kg did not suppress plasma erythropoietin elevated by any stimuli. Repeated injection of diethylstilbestrol at 1,000 μg/kg to rats for 1 month induced an anemic trend due to decelerated erythropoiesis through the insufficient production of erythropoietin, mimicking the effects of estradiol. In conclusion, diethylstilbestrol has a suppressive effect on erythropoietin induction, leading to deceleration of erythropoiesis and the development of anemia.Archives of Toxicology 07/2013; · 5.22 Impact Factor
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ABSTRACT: In mammals, estrogens have been described as endocrine and paracrine modulators of neuronal differentiation and synapse formation. However, the functional role of circulating estrogens and the distribution of estrogen receptors (ERs) in the cerebral cortex of reptiles have not been clearly established. Caiman latirostris (C. latirostris) is a South American species that presents temperature-dependent sex determination (TSD). By using immunohistochemistry, we have studied the distribution of ERα in the cerebral cortex of neonatal caimans. We studied brain samples from ten-day-old TSD-females and TSD-males and from female caimans that were administered estradiol during embryonic development (hormone-dependent sex determination, HSD-females). ERα was detected in the medial (MC), dorsal (DC) and lateral (LC) cortices. ERα expression in the MC showed sex-associated differences, being significantly greater in TSD-females compared to TSD-males. Interestingly, the highest ERα expression in the MC was exhibited by HSD-females. In addition, the circulating levels of estradiol were significantly higher in females (both TSD and HSD) than in TSD-males. Double immunostaining showed that ERα is expressed by neural precursor cells (as detected by ERα/doublecortin or ERα/glial fibrillary acidic protein) and mature neurons (ERα/neuron-specific nuclear protein). Our results demonstrate that the expression of ERα in the neonatal caiman cortex is sexually dimorphic and is present in the early stages of neuronal differentiation.General and Comparative Endocrinology 08/2012; 179(2):205-13. · 2.82 Impact Factor
Brain Research Bulletin 71 (2007) 619–627
Diethylstilbestrol alters the population dynamic of neural precursor
cells in the neonatal male rat dentate gyrus
Jorge G. Ramos∗, Jorgelina Varayoud, Lucas Monje, Guillermo Moreno-Piovano,
M´ onica Mu˜ noz-de-Toro, Enrique H. Luque
Laboratorio de Endocrinolog´ ıa y Tumores Hormonodependientes, School of Biochemistry and Biological Sciences,
Universidad Nacional del Litoral, Casilla de Correo 242, 3000 Santa Fe, Argentina
Received 16 September 2006; received in revised form 21 November 2006; accepted 5 December 2006
Available online 4 January 2007
Little is known about how estrogens influence neurogenesis in the newborn male rodent. Herein, we examined the effects of neonatal diethyl-
stilbestrol (DES) exposure on the proliferation and survival of type-1 and type-2 neural precursor cells (NPC) in the dentate gyrus of male rats.
This was achieved by exposing newborn male pups to DES on postnatal day (PND) 1, PND3, PND5, and PND7, sacrificed at PND8 or PND21,
followed by double immunohistochemistry and morphometric analysis of hippocampal dentate gyrus. Furthermore, vascular endothelial growth
factor (VEGF) and brain-derived neurotrophic factor (BDNF) mRNA expression was evaluated in hippocampal tissue blocks by real time RT-PCR.
At PND8, the density of total proliferating NPC decreased in DES-treated animals. This reduction was due to a significant decrease in the mitotic
rate of type-2 cells only, since type-1 NPCs did not show changes in the proliferation index. Type-2 NPCs expressed the cell-cycle inhibitor p27kip1
and its expression was clearly augmented in the DES-treated group. Furthermore, the number of apoptotic cells in the dentate gyrus of DES-treated
rats decreased. Surprisingly, DES treatment enhanced cell survival and increased NPCs proliferation when animals were examined 14 days after
treatment. VEGF mRNA expression showed a positive correlation with NPCs proliferation and BDNF mRNA levels were higher in DES-treated
animals at both time points examined. Collectively, these results indicate that hippocampal NPCs proliferation and survival is a critical target of
DES exposure during the early postnatal period. VEGF and BDNF are proposed as key mediators of DES-induced NPC mitotic response.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Hippocampus; Neural precursors; DES; Proliferation; Apoptosis
Neurogenesis occurs throughout life in mammals includ-
ing man [14,23]. Neural precursor cells (NPC), located in the
subgranular zone (SGZ) of the dentate gyrus, proliferate and
forms of hippocampal-dependent learning [19,47]. Two NPC
subtypes (type-I and type-II NPCs) have been characterized
previously using transgenic mice expressing green fluorescent
∗Corresponding author. Tel.: +54 342 4575207; fax: +54 342 4575207.
E-mail address: firstname.lastname@example.org (J.G. Ramos).
and reach the inner most portion of the molecular zone while
type-II NPCs lack these long processes and are characterized
by short cytoplasmic extensions that are tangentially oriented
to the GCL . Under resting conditions, type-II NPCs are
most actively dividing, and physiologic mitogenic stimuli seem
to affect primarily these early types of precursors. Type-I cells
rarely divide, and their proliferation is not induced by the exper-
imental manipulations studied so far [13,26].
Previous studies have identified endocrine, neural, and
experiential factors that regulate the production and survival
of late-generated neurons in the rat hippocampus [29,35].
These include negative modulators, such as glucocorticoids
and interleukin-6 [34,51], and positive modulators, such as
serotonin, N-methyl-aspartate receptor antagonists and dehy-
Estrogens have been shown as regulators of synaptic plas-
ticity, cognitive function, memory, mood and behavior ;
however, their roles in the control of neuronal and glial prolif-
0361-9230/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
J.G. Ramos et al. / Brain Research Bulletin 71 (2007) 619–627
eration and differentiation are still not fully understood. Recent
reports have shown that estradiol is able to influence cell pro-
duction in the adult hippocampal formation [41,50]. A single
dose of estradiol (10?g/rat) has been shown to be capable
of increasing NPC proliferation in the SGZ of ovariectomized
adult female rats . In contrast, other studies have demon-
strated that the density of new cells is negatively correlated
with plasma estradiol levels in wild adult female meadow voles
and laboratory rats [37,38]. This apparent discrepancy could
be explained by a time- and dose-dependent action of estro-
gen on NPC proliferation [37,38,50]. Moreover, the positive or
negative action of estrogens on hippocampal cell proliferation
Some efforts have been taken to elucidate possible mecha-
nisms and molecular mediators underlying NPC proliferation
and differentiation. Vascular endothelial growth factor (VEGF)
has been proposed as a key molecule that could regulate neuro-
genesis, mediating the effects of environment on hippocampal
plasticity during adulthood . Most research on the effects of
estrogens on neurogenesis has been performed on adult female
rodents, and thus, little is known about the consequences of
estrogen exposure on hippocampal NPCs proliferation in the
neonatal male brain. There is a great body of evidence prov-
ing that the perinatal period is critical on hippocampal circuitry
formation . During development, estrogen treatment has
been shown to affect the volume of CA1 and CA3 hippocam-
pal regions, spatial navigation behavior , the strength of
hippocampal long-term potentiation [8,57], and hippocampal-
dependent learning . The GCL of the hippocampal dentate
gyrus has been characterized as a region of continuous but age-
dependent proliferation of NPC in rodents and humans [14,56].
DNA labeling studies performed in rats have demonstrated that
NPCs proliferation is intense during the first postnatal week
in the SGZ of the dentate gyrus, declining from the second
postnatal week to adulthood [18,44].
DES is a synthetic estrogen that is frequently used in neona-
tal estrogenization experiments because it is not bound by
alpha-fetoprotein and penetrate the blood brain barrier [12,46].
Previously, it has been shown that DES exposure during the
developmental stage resulted in a marked influence on synapto-
genesis and neuronal vulnerability . Several reports have
indicated that neonatal xenoestrogen exposure inhibits sex-
ual differentiation of non-reproductive behaviors, including
hippocampal-dependent tasks, like maze learning and spatial
location [4,15]. However, very little is known about the effects
and mechanisms underlying DES exposure during the early
postnatal period on the male hippocampal NPC population
Taking into account the above-mentioned findings, the goal
of the present study was to determine the effects of DES expo-
sure on the proliferation, apoptosis and survival of hippocampal
NPC in vivo, during the first week of male rat postnatal life.
To evaluate the possible molecular mediators of these effects, a
real time RT-PCR approach was optimized to quantify VEGF
and BDNF mRNA expression levels in the dorsal dentate gyri
of neonatally estrogenized male rats.
2. Materials and methods
2.1. Animal treatments
Pups were obtained from timed-pregnant Wistar rats housed under a con-
supplied from glass bottles. All rats were handled in accordance with the prin-
ciples and procedures outlined in the Guide for the Care and Use of Laboratory
Animals issued by the National Academy of Sciences (USA). All protocols
were approved by the Bioethical Commission of the School of Biochemistry
and Biological Science of the Universidad Nacional del Litoral.
Seven pregnant dams per treatment group were used to collect offspring for
each time point (see below). The day of birth was designated as postnatal day
0 (PND0). Pups were sexed according to ano-genital distance, and each litter
was culled randomly on PND1 to a maximum of 10 neonates, with up to 10
male pups per litter if possible. When fewer than 10 males were available, an
appropriate number of females were retained. All male pups were assigned to
one of two groups, and were given sc injections of either DES (0.2?g/pup,
Sigma, St. Louis, MO) dissolved in corn oil or an equal volume (25?l) of corn
oil alone on PND1, PND3, PND5, and PND7.
The DES dose used here did not produce signs of acute or chronic toxicity
and no significant differences in weight gain between DES-exposed and control
pups were recorded during the experiment (data not shown). On PND8, a set
of males of both groups was injected i.p. with the thymidine analog bromod-
eoxyuridine (BrdU, Sigma, 60mg/kg of body weight). To determine the NPC
proliferation index on PND8, some animals (n=12 for each group) were killed
by decapitation 4h after BrdU injection. To evaluate the NPC survival index,
others were sacrificed 14 days (PND21) after BrdU treatment (n=10 for each
group). To assess NPC proliferation 2 weeks after DES treatment, another set of
rats was injected with BrdU on PND21 and sacrificed 4h later (n=13 for each
group). For immunohistochemical procedures, brains from males sacrificed at
brains (n=10 per treatment group in each time point) was quickly removed, and
the dorsal pole of the hippocampus (including the dentate gyrus) was microdis-
sected, using needles of adequate internal diameters . Tissue blocks were
flash frozen in liquid nitrogen and stored at −80◦C until RNA extraction.
Brain serial sections (5?m in thickness) were mounted on 3-aminopropyl
triethoxysilane (Sigma) coated slides and dried for 24h at 37◦C. BrdU incor-
poration into cells in the S phase of the cell cycle was evaluated as previously
described . All primary antibodies were incubated overnight at 4◦C. For
BrdU labeling, a mouse monoclonal antibody against BrdU (clone 85-2C8,
Novocastra, Newcastle upon Tyne, UK) was used at a dilution of 1:100.
Immunostaining of estrogen receptor ? (ER?) was performed using a mouse
monoclonal antibody raised to full-length recombinant human ER? (clone
6F11, Novocastra) at a dilution of 1:200, and for the detection of p27kip1,
a polyclonal antibody diluted 1:500 raised against a peptide mapping at the
carboxy terminus of p27 of human origin was evaluated (sc-528, Santa Cruz
Biotechnology Inc., CA). Reactions were developed using a streptavidin–biotin
peroxidase method and diaminobenzidine (DAB) (Sigma) as a chromogen sub-
strate. Samples were counterstained with Harris hematoxylin (Biopur, Rosario,
Argentina) and mounted with permanent mounting medium (PMyR, Buenos
Aires, Argentina). Each immunohistochemical run included positive and nega-
tive controls. For negative controls, the primary antibodies were replaced with
non-immune mouse or rabbit serum (Sigma), or immunohistochemistry (IHC)
was performed in samples from animals that did not receive BrdU.
2.3. Double IHC
To identify the immunophenotype of the proliferating or quiescent cells, a
using anti-nestin (identifying type-1 and type-2 NPCs) , anti-calbindin (to
detect mature granule neurons), or anti-GFAP (to detect astrocytes) as a second
J.G. Ramos et al. / Brain Research Bulletin 71 (2007) 619–627
(clone rat 401, BD Pharmingen, San Jose, CA) 1:200, or anti-calbindin-D-
28-K (KD-15, Sigma) 1:200, or anti-GFAP (Sigma) 1:100. Reactions were
developed using a streptavidin–biotin peroxidase method. Visualization of this
The DAB-nickel solution (2.3mg DAB, 4ml 0.05M Tris–HCl buffer [pH 7.5],
15?l 30% H2O2, and 460?l 1% nickel chloride) was added to the samples.
After 10min at 20◦C, the samples were rinsed in running water. Slides were
counterstained with Harris hematoxylin (Biopur) and mounted with permanent
2.4. In situ detection of apoptosis
Sections were analyzed for in situ detection of cells with DNA strand breaks
(apoptotic cells) using the TUNEL technique (ApopTag, Intergen Co., Pur-
chase, NY). In brief, after deparaffinization and rehydration, sections were
incubated with proteinase K (20?g/ml) (Intergen) for 15min at 37◦C and then
treated with hydrogen peroxide in PBS for 10min at RT to quench endogenous
peroxidase activity. Sections were then incubated with a mixture containing
digoxigenin-labeled deoxynucleotide triphosphate, unlabeled deoxynucleotide
triphosphate mix, and TdT enzyme in a humidified chamber at 37◦C for 1h.
Slides were subsequently rinsed with PBS and incubated with anti-digoxigenin-
for 6min. Samples were counterstained with Mayer hematoxylin, dehydrated,
and mounted with a permanent mounting medium. Negative control slides were
ran using the same procedures, except that distilled water was added instead of
TdT enzyme. As a positive control, involuting rat prostate after the second day
of castration was processed in an identical manner to the experimental samples.
To avoid interference of possible neurogenetic gradients  in differences
depending on the treatment, the same hippocampal region was analyzed in all
groups for BrdU and neuronal markers immunohistochemistry and TUNEL
staining. The area of the hippocampus evaluated in this work was limited to
the dorsomedial pole of the hippocampus, where the dentate gyrus is horizon-
tally oriented beneath the corpus callosum and the external and internal blades
are joined at the crest (an approximate 0.8mm block of tissue extending in the
caudal direction from the first appearance of the GCL) [6,27,40]. The SGZ was
defined as a 150?m band immediately adjacent to the hilar surface of the GCL.
The cell counting procedures were performed in the GCL and the SGZ of the
dentate gyrus as was previously described [6,40]. To calculate the proportions
of each cell phenotype and the density of BrdU-positive cells in the dentate
gyrus, the total number of BrdU or p27kip1-labeled cells and their respective
phenotypes were scored in six or more sections of a one-in-six series per ani-
mal. To evaluate the entire dorsomedial pole of the hippocampus the different
series of coronal sections were separated in the caudal direction by approxi-
mately 120?m. The ER?(+) cell population and the proportion of apoptotic
cells were evaluated using the same criteria. All data were represented as spatial
cell densities (number of cells/mm2of evaluated area) or relative proportions.
All spatial measurements (SGZ and GCL area and volume) were performed
using an Olympus BH-2 microscope equipped with a Spot Insight color video
MD). To calculate the total volumes of the portions of the GCL and SGZ ana-
lyzed, two consecutive sections every 120?m were selected and the outlines of
the GCL and the SGZ were drawn with a low-power objective lens (10×). The
estimation of GCL and SGZ volumes were made according to the principle of
Cavalieri . The areas were calculated by means of Image Pro-Plus software
and the total volume for each hemisphere was estimated as the sum of the areas
multiplied by the distance between the analyzed sections.
2.6. Real-time PCR analysis
sion levels of VEGF and BDNF mRNAs in the dentate gyrus of male rats on
PND8 and PND21. Individual samples of hippocampal dentate gyri of each
experimental group were homogenized in TRIzol (Invitrogen, Carlsbad, CA),
and RNA was prepared according to the manufacturer’s protocol. Equal quan-
tities (4?g) of total RNA were reverse-transcribed into cDNA with AMV
primers (Promega). Twenty units of ribonuclease inhibitor (RNAout) (Invitro-
gen Argentina, Buenos Aires, Argentina) and 100nmol of a deoxynucleotide
triphosphate mixture were added to each reaction tube in a final volume of 30?l
Reactions were stopped by heating at 97◦C for 5min and cooling on ice, fol-
lowed by dilution of the reverse-transcribed cDNA with RNAse free water to a
final volume of 60?l. Samples were analyzed in duplicate or triplicate, and a
ination by genomic DNA. Primer pairs used for amplification of VEGF, BDNF
and the ribosomal protein L19 cDNAs are shown in Table 1. cDNA levels were
detected using real-time PCR with the DNA Engine Opticon System (Bio-Rad
Laboratories Inc. Waltham, MA) and SYBR Green I dye (Cambrex Corp., East
Rutherford, NJ). For cDNA amplification, 5?l of cDNA were combined with
a mixture containing 2.5U Taq-DNA polymerase (Invitrogen), 2.5mM MgCl2
(Invitrogen), 0.2mM of each of the four dNTPs (Promega), and 10pmol of each
primer (Invitrogen) in a final volume of 25?l of 1× SYBR Green I PCR Taq
jected to successive cycles of denaturation at 96◦C for 45s, annealing at 60◦C
for VEGF and BDNF genes for 30s, and extension at 72◦C for 1min. The
annealing temperature for the amplification of the L19 housekeeping gene was
gel electrophoresis. No DNA template controls were included in any of the
assays, yielding no consistent amplification. Calculation of relative expression
levels of each target was conducted based on the cycle threshold (CT) method
. The CTfor each sample was calculated using the Opticon Monitor Anal-
ysis Software (Bio-Rad Laboratories) with an automatic fluorescence threshold
setting. Efficiency of PCR reactions was assessed for each target by amplifica-
transcripts under analysis. Depending on specific PCR conditions, efficiencies
subtracting the corresponding L19 CTvalue (internal control) from the specific
CTof each target and experimental condition. ??CTis obtained by subtracting
the ?CTof each experimental sample from that of the control sample (taken
as reference value 100). Note that no significant differences in CTvalues were
observed for L19 between the treatment groups. All PCR products were cloned
using the TA cloning kit (Invitrogen, Argentina) and specificity was confirmed
by DNA sequencing (data not shown).
Primer pairs used in real-time RT-PCR
GenePrimer sequence (5?–3?)Product size (bp) Genbank accession number
Forward: AGCCTCCTCTG TCTTTCTGCTGGA
J.G. Ramos et al. / Brain Research Bulletin 71 (2007) 619–627
Values are expressed as mean±S.E.M.; and comparisons were made using
the Mann–Whitney non-parametric test and the threshold of significance was
fixed at P<0.05.
3.1. Neonatal xenoestrogen exposure alters the NPC
3.1.1. NPC proliferation and apoptosis on PND8
Proliferating cells were detected in the molecular layer,
hilus, SGZ and in the GCL innermost portion of the den-
tate gyrus in controls and DES-treated rats. They were often
organized in clusters, with irregularly shaped nuclei. In the
dentate gyrus of control PND8 rats, there were more BrdU-
positive cells than in age-matched DES-treated rats (Fig. 1,
A versus B). Morphometric analysis demonstrated that treat-
ment with DES decreased cell proliferation (P<0.01, Fig. 2A).
As described previously , NPCs are characterized by mor-
phological features and by the expression of the filamentous
protein nestin. Nestin-expressing precursor cells fall into two
large categories: type-1 and type-2 NPCs. Type-1 NPC is gener-
ally located in the SGZ and characterized by a long process
(Fig. 1C, inset C1) reaching through the GCL and into the
molecular layer of the dentate gyrus. Type-2 NPC lacks long
Fig. 1. Effects of neonatal DES exposure on the NPC population dynamic in male pups at PND8. Male pups were injected from PND1 to PND7 with either vehicle
(A, C, E and G) or 0.2?g of DES (B, D, F and H). BrdU(+) cells were diminished in the dentate gyrus of PND8 DES-treated male rats (A vs. B). The two main
categories of nestin-expressing cells were morphologically characterized in the SGZ: type-1 (C1 inset arrows) and type-2 (C2, inset arrows) NPCs; only type-2 NPC
proliferation index was decreased in DES-treated animals (C vs. D, arrowheads). However, the number of apoptotic cells (E, arrows) was decreased in DES-treated
animals (E vs. F). Expression of p27kip1was only detected in type-2 NPC (G and H, arrows) and was increased in DES-treated group (G vs. H). GCL, granular cell
layer; SGZ, subgranular zone. Scale bar 100?m.
J.G. Ramos et al. / Brain Research Bulletin 71 (2007) 619–627
Fig. 2. Quantification of the results obtained by TUNEL technique and by regular and double immunohistochemistry in the dentate gyrus on PND8. Data are
expressed as spatial cell densities and each column represents the mean±S.E.M. of atleast four semi-serial sections per animal (n=12 animals per group). (A) Total
proliferating cells in the SGZ; (B) total NPC proliferation density; (C) NPC types-I and -II proliferation indexes; (D) apoptotic cells; (E) total NPC p27kip1(+) cells;
(F) NPC type-I and type-II p27kip1(+) density. Scale bar 100?m.
processes having a round or ovoid nucleus and a soma with
scant cytoplasm (Fig. 1C, inset C2). The short cytoplasmic
extensions of these cells tended to be oriented tangentially
to the GCL. Using double IHC, we detected that type-1 and
type-2 cells had incorporated BrdU 4h after i.p. administra-
tion, indicating that both cell types had undergone proliferation
in controls and in DES-treated males. On PND8, the density
of total BrdU(+)/nestin(+) NPC decreased in DES-treated ani-
mals (P<0.01, Fig. 1, C versus D and Fig. 2B) however, when
BrdU(+)/nestin(+) cells were evaluated identifying each NPC
type, the decrease in proliferative activity was due to type-
2 cells only (P<0.01, Fig. 2C). On the same postnatal day,
the number of pyknotic and TUNEL(+) cells in the SGZ of
DES-treated rats clearly decreased (P<0.01, Fig. 1, E ver-
sus F, and Fig. 2D), suggesting an anti-apoptotic effect of the
xenoestrogen exposure during the early postnatal period. In
accordance with the above-mentioned findings, an increased
expression of p27kip1was found in the SGZ of DES pups
(Fig. 2E). By double IHC, we could classify which type of
NPC express p27kip1, showing that only type-2 NPC expresses
this cell-cycle inhibitor and its expression was clearly aug-
mented in DES-treated group (P<0.01, Fig. 1, G versus H and
3.1.2. NPC survival on PND21
ined the number of BrdU(+) cells that remains in the SGZ and
of the BrdU-labeled cells in both groups was fully incorporated
into the GCL and they were morphologically similar to the sur-
rounding mature granule neurons. Surprisingly, the number of
controls (P<0.05, Fig. 3A). Regarding the immunophenotype
of the surviving BrdU(+) cells, results showed that DES did
not alter the proportion of calbindin (mature neuron type) or
GFAP (mature glial type) positive cells compared with controls
3.1.3. NPC proliferation on PND21
In contrast with the results observed on PND8, DES-treated
rats that were injected with BrdU on PND21 showed a sig-
nificantly higher number of double labeled BrdU(+)/nestin(+)
cells compared with control rats (P<0.01, Fig. 4A). In addi-
tion, the NPC of DES-treated group showed a clearly decreased
expression of p27kip1(P<0.01, Fig. 4B). At this time point, no
differences in the number of apoptotic figures were observed
between the controls and DES-treated males (Fig. 4C).
J.G. Ramos et al. / Brain Research Bulletin 71 (2007) 619–627
Quantitative analysis of GCL and SGZ volumes
0.351 ± 0.081
0.732 ± 0.021
0.151 ± 0.009
0.387 ± 0.012
0.339 ± 0.015
0.799 ± 0.056
0.148 ± 0.008
0.401 ± 0.022
Results are expressed in mm3as means±S.E.M.
3.1.4. ERα expression in NPCs
Finally, no differences were found in the number or distribution
of total ER?(+) cells in both experimental groups at any time
point analyzed (data not shown).
3.1.5. GCL and SGZ volumes
The estimated volumes of the GCL and SGZ analyzed in this
work are shown in Table 2. No differences were obtained in the
volumetric analysis between DES treated and control animals.
3.2. Hippocampal VEGF and BDNF gene expressions are
affected by neonatal DES treatment
Using quantitative real time RT-PCR we examined the
expression of VEGF and BDNF mRNA in the microdis-
Fig. 3. Two weeks after DES exposure, animals showed a higher number of
surviving BrdU(+) cells in the dentate gyrus. (A) Spatial density of BrdU(+)
cells and morphological detail of surviving cells in each experimental group
(arrows) and (B) proportion of BrdU(+) surviving cells that show neuron or
glial phenotype at PND21. (*P<0.05 vs. control).
sected dentate gyrus of experimental males on PND8 and
PND21. Results demonstrated that DES treatment significantly
decreased the VEGF mRNA expression in the hippocampus of
PND8 rats (P<0.01, Fig. 5A). In contrast, 14 days after the
end of the xenoestrogen regime (PND21), VEGF mRNA lev-
els were clearly augmented in DES-treated animals (P<0.01,
Fig. 5A). Additionally, BDNF mRNA levels were higher in
DES-treated animals at both times points when compared with
control animals (P<0.01, Fig. 5B).
PND21. BrdU was injected 4h before sacrifice on PND21. Data are expressed
as spatial cell densities and each column represents the mean±S.E.M. of five
semi-serial sections per animal (n=13 animals per group). (A) PND21 NPC
proliferation; (B) PND21 NPC p27kip1(+) density; (C) PND21 apoptotic cells
density. (*P<0.05 vs. control).
J.G. Ramos et al. / Brain Research Bulletin 71 (2007) 619–627
Fig. 5. Effect of neonatal DES exposure on (A) VEGF mRNA and (B) BDNF
mRNA levels in the rat dentate gyrus on PND8 and PND21. Relative mRNA
levels were measured by real-time RT-PCR and fold expression from control
values were calculated for each target by the equation 2−??CT. Control values
were assigned to a reference level of 100 and values are given as mean±S.E.M.
of atleast three independent determinations. (*P<0.05 vs. control).
To our knowledge, this is the first study showing that DES
reduction in the proliferative activity of hippocampal NPCs was
observed the day after the end of DES treatment while a signifi-
2 weeks after the last DES injection. This biphasic NPC prolif-
eration response positively correlated with hippocampal VEGF
mRNA levels, suggests that VEGF could be a key mediator in
this estrogen-induced event. Taking into account that the prolif-
tempting to propose that the observed changes in the hippocam-
pal cell proliferation were due to DES-induced modifications in
type-2 NPCs. Moreover, p27kip1was only expressed in type-2
cells and was negatively correlated with BrdU immunodetec-
tion on PND8 and PND21. Collectively, these results show that
type-2 NPC population constitutes a target of DES action.
Many studies have described that estrogen could affect cell
et al.  have shown that an acute treatment with a moderate
(10?g/rat), but not with a low (1?g/rat) or a high (50?g/rat),
dose of 17?-estradiol rapidly increased cell proliferation in
ovariectomized adult animals. Moreover, no differences were
observed when the steroid was given during prolonged peri-
ods of time (3 weeks). These results clearly showed that the
effects of estrogens in the control of cell proliferation on the
dentate gyrus of adult female rats depended on the selected
times and doses. However, little is known about the estrogen
effects on dentate gyrus cell proliferation during the early post-
natal period. Our results show that the DES treatment used was
enough to alter the NPCs population dynamic. At PND8, a clear
down-regulation of type-2 NPC proliferation was observed in
the DES-treated males, suggesting that the early postnatal time
between PND8 versus PND21 and those obtained by others in
adult rats [49,50] suggest that the estrogenic responsiveness of
the NPCs vary at different life-time points. This observation is
important since a growing body of evidence shows that man and
wildlife are exposed to estrogenic active compounds during the
perinatal period . The behavioral and physiological conse-
quences of an estrogenic perturbation on NPC proliferation and
differentiation during early postnatal times are still unknown.
Many reports have shown that perinatal exposure to estrogen-
like compounds alters exploratory and socio-sexual behavior
[17,43]. Future studies could address whether these estrogen-
induced behavioral disorders are related with perturbations in
the proliferation and survival of hippocampal NPCs.
Several reports have been demonstrated the importance of
a functional septohippocampal pathway for the regulation of
hippocampal neurogenesis [7,33,52]. There is a growing body
of evidence proving that the cholinergic basal forebrain sys-
tem plays a survival-promoting role for neuronal progenitors
and immature neurons within regions of adult neurogenesis
like the SGZ of the dentate gyrus . It has been clearly
tral role in the modulation of hippocampal Ach release .
Since the cholinergic forebrain neurons activity is regulated by
estrogens through ER?  an indirect cholinergic pathway in
DES-mediated control of hippocampal neurogenesis could not
be ruled out.
marily on the duration of the G1 transition and is modulated by
the enzymatic activity of cyclin-dependent kinases (CDKs) .
It has been shown that CDK regulators, like the kip1 protein
family, may have a critical role in cell-cycle control of transit
mice brain . During embryonic development, it has been
shown that p27kip1is implicated in the maintenance of differen-
the possible roles of this CDK inhibitor during postnatal neu-
rogenesis . Using double IHC together with morphological
criteria, we demonstrated that type-2 NPCs express p27kip1pro-
tein and this expression were enhanced with DES treatment. An
was observed, suggesting a critical role of this CDK inhibitor in
xenoestrogen-induced quiescent state of type-2 NPCs. Not only
a great number of type-2 NPCs remained quiescent in DES-
treated males on PND8 but a significant decrease in the number
could contribute to the great proportion of remaining BrdU(+)
cells observed on PND21 in the DES group, strongly suggest-
ing that xenoestrogen exposure enhances the survival of cell
progeny in the male dentate gyrus, while no effects on NPC
J.G. Ramos et al. / Brain Research Bulletin 71 (2007) 619–627
differentiation was detected. Moreover, when BrdU incorpora-
tion was evaluated on PND21, a great number of NPCs were
positive in the DES-treated group, suggesting the existence of
an internal regulatory mechanism triggered to compensate the
initial xenoestrogen-induced declination in NPCs proliferation.
There is a growing body of evidence that suggest that extrin-
sic agents, such as alcohol or xenoestrogens can alter intrinsic
cellular mechanisms of stem cell fate choices contributing to
altered neurogenesis and/or gliogenesis during central nervous
system maturation . The compensatory mechanism above-
mentioned could help to restore the number of required granule
early estrogenic exposure. Whether this mechanism is inducible
is under study in our laboratory.
VEGF is a hypoxia-inducible protein that promotes angio-
genesis through receptor tyrosine kinases on endothelial cells.
Recent evidence indicates that VEGF can act as a neurotrophic
factor [31,36] and produces neurogenic effects on NPCs .
A significant reduction in hippocampal VEGF transcripts was
observed on PND8 in DES-treated rats in parallel with a reduc-
tion in NPCs proliferation. On the other hand, on PND21, when
the NPCs proliferation was higher in the estrogenized-rats the
VEGF mRNA levels were significantly increased. We propose
here that hippocampal VEGF could be a xenoestrogen target
gene and a positive molecular mediator in type-2 NPC prolifer-
Regarding the role of BDNF, it has been shown that estrogen
could up-regulate the BDNF mRNA and protein levels in the rat
hippocampus . Our results agree with this study, since we
observed a significant increase in BDNF mRNA expression in
and plays an important role in the survival and growth of neu-
rons . However, the possible relationships between BDNF
and NPCs proliferation remain unknown. Taking into account
that we observed a decrease in apoptotic levels and an increase
not be ruled out. We also suggest that VEGF is a key factor in
type-2 NPC regulation of proliferation, and that BDNF could
be responsible for extended survival times of the neuronal cell
progeny. More studies are necessary to confirm this hypothesis.
Conflict of interest
The authors declare that there is no conflict of interest that
would prejudice the impartiality of this scientific work.
The authors thank Mr. Juan C. Villarreal and Mr. Juan Grant
for their technical assistance and animal care. This study was
supported by grants from the Argentine National Council for
Science and Technology (CONICET, CIC Grant 652/04), the
Argentine National Agency for the Promotion of Science and
Technology (ANPCyT) (PICT 2003, No. 13-4737) and the Uni-
versidad Nacional del Litoral (CAI+D 2005 019/118). L.M.
and G.M.P. are fellows of the CONICET and the Universidad
Nacional del Litoral, respectively. J.V., J.G.R., and E.H.L. are
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