Human Molecular Genetics, 2013, Vol. 22, No.

6 1086–1096
doi:10.1093/hmg/dds510
Advance Access published on December 5, 2012

Developmental abnormalities in mouse embryos

lacking the HDL receptor SR-BI
Nicolás Guillermo Santander1, Susana Contreras-Duarte1, Marı́a Fernanda Awad1,
Carlos Lizama1, Isabella Passalacqua1, Attilio Rigotti1,2 and Dolores Busso1,∗
1
Departamento de Nutrición, Diabetes y Metabolismo, Pontificia Universidad Católica de Chile, Santiago, Chile and
2
Departamento de Gastroenterologı́a. Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile

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Received September 27, 2012; Revised November 20, 2012; Accepted November 28, 2012

The srbi gene encodes a lipoprotein receptor with high affinity for high density lipoprotein that is mainly
expressed in the liver and in steroidogenic tissues. Disruption of this gene in mice and mutations
in humans lead to alterations in lipoprotein metabolism and/or fertility. During murine development, scav-
enger receptor class B member I (SR-BI) is present in the yolk sac and the placenta and is only expressed
in the embryo itself late in gestation. In humans, it has been detected in trophoblast cells and placenta.
Although the proportion of mice carrying a null mutation in SR-BI obtained from heterozygous inter-
crosses is lower than the expected by the Mendelian ratio, suggesting the involvement of this receptor
in intrauterine development, the cause of this demise has remained unknown. In this work, we show
that embryos lacking SR-BI exhibit a high prevalence of exencephaly with a sex bias toward females.
Immunolocalization studies confirmed that SR-BI is not expressed in the embryo at early stages of devel-
opment and allowed a more detailed description of its localization in the cells that mediate maternal –fetal
transport of nutrients. SR-BI-null embryos contain less cholesterol than their wild-type littermates, sug-
gesting the involvement of SR-BI in materno-fetal cholesterol transport. Newborn SR-BI-deficient pups
exhibit intrauterine growth restriction, suggesting that this receptor is also important for fetal growth.
Altogether, the results of our work suggest that the presence of SR-BI in extraembryonic tissues is
involved in the maternal – fetal transport of cholesterol and/or other lipids with a role during neural tube
closure and fetal growth.

INTRODUCTION phenotypes related to the defective lipoprotein metabolism, in-

The scavenger receptor class B member I (SR-BI) is a multi-
ligand protein that binds lipoproteins, glycoproteins and
anionic phospholipids. It binds high density lipoprotein
(HDL) with high affinity allowing bidirectional transport of
liposoluble molecules to and from lipoprotein particles and
is a key regulator of plasma cholesterol levels in the adult
mouse (1 – 3). In the liver, SR-BI mediates the uptake of cho-
lesteryl esters from cholesterol-rich HDL particles for its ex-
cretion to the bile, and in steroidogenic tissues, this receptor
provides cholesterol for steroid hormones synthesis. Mice car-
rying a null mutation in the srbi locus exhibit an accumulation
of cholesterol in abnormal HDL particles and different
cluding high susceptibility to atherosclerosis, adrenal insuffi-
ciency and female infertility (3). No loss-of-function
mutations have been reported for the gene encoding SR-BI
in humans, in contrast to the gene for the low density lipopro-
tein LDL receptor (LDLR), where mutations induce hyper-
cholesterolemia due to impaired LDL endocytosis. However,
recent studies have shown that an abnormal SR-BI expression
and/or function can also affect human lipoprotein metabolism
and health (4,5). An interesting observation provided by the
genetic inactivation of SR-BI in mice was that the proportion
of knockout animals weaned from heterozygous intercrosses
was lower than expected according to the Mendelian ratio,
leading researchers to postulate for years that the lack of

∗
To whom correspondence should be addressed at: Departamento de Nutrición, Diabetes y Metabolismo, Escuela de Medicina, Pontificia Universidad
Católica. Marcoleta #367, interior, 4to Piso, Santiago, Chile. Tel: +56 (2)23543841; Email: dbusso@med.puc.cl

# The Author 2012. Published by Oxford University Press. All rights reserved.
For Permissions, please email: journals.permissions@oup.com

SR-BI could hinder embryo development (2). In mice, SR-BI
is dynamically expressed in the maternal – fetal interfaces
during pregnancy (6). Interestingly, SR-BI2/2 E16.5
fetuses have been shown to take up less radioactive cholesterol
than their wild-type and heterozygous littermates (7). In
humans, SR-BI is expressed in term placenta, and its protein
levels are reduced in intrauterine growth restriction (IUGR)
when compared with normal pregnancies (8). Furthermore,
human fetal placental endothelial cells express SR-BI and
are able to efflux cholesterol to HDL particles (9).
Several lines of evidence show that cholesterol is necessary
for normal embryo development in mammals. In humans, the
most common inborn defect in cholesterol metabolism is the
Smith– Lemli –Opitz (SLO) syndrome, caused by mutations
in 7-dehydrocholesterol reductase (7-DHCR), the enzyme
that catalyzes the last step in cholesterol synthesis. This
disease exhibits several clinical manifestations, ranging from
mental retardation to the lethal cranial malformation holopro-
cencephaly (10,11). Interestingly, SLO infants with null muta-
tions in 7-DHCR still show measurable amounts of cholesterol
indicating that there is provision of this lipid from maternal
sources (12). Although the relative contribution of cholesterol
from endogenous synthesis versus maternal source for embry-
onic/fetal development is still controversial, the direct uptake
of maternal cholesterol by fetuses has been demonstrated in
different species, including mice and humans (13,14).
The extraembryonic tissues responsible for the absorption
of lipids from the maternal bloodstream express numerous
proteins involved in lipoprotein metabolism, including very
low density lipoprotein receptor, LDLR, ApoER1/2, LDL
receptor-related protein (LRP1/2), cubilin, ABCA1 and, as
mentioned previously, SR-BI (6,15– 17). Moreover, the pla-
centa and yolk sac in mice as well as the human placenta
mediate the regulated transport of lipoprotein lipids during
post implantation development (12,13,18). Mouse mutants de-
ficient in different proteins involved in lipoprotein metabol-
ism, i.e. cubilin (19), LRP1 (20), LRP2/Megalin (21), ApoB
and microsomal triglyceride transfer protein (22 – 24) have
shown developmental abnormalities. At present, no embryonic
defects have been associated with SR-BI inactivation.
Based on previous evidence showing that SR-BI is expressed
in tissues from the maternal – fetal interfaces and its deficiency is
associated with the demise of SR-BI2/2 mice, we hypothe-
sized that the lack of SR-BI-mediated cholesterol transport
hindered an essential developmental process that affected
the viability of SR-BI-deficient mouse embryos. The aim of
this study was to evaluate the existence of possible phenotype(s)
leading to lethality in SR-BI2/2 mouse embryos and to
analyze the participation of SR-BI in materno-fetal cholesterol
transport using this model. First, homozygous null (SR-
BI2/2) mice born from heterozygous (SR-BI+/2) progeni-
tors were retrieved at three stages of gestation and at birth and
were then analyzed to evaluate possible lethality-inducing ab-
normalities. Next, the presence of SR-BI in embryos and extra-
embryonic tissues at those developmental stages was
determined by immunohistochemistry. Finally, whole body
cholesterol content was compared between SR-BI2/2 and
their littermates to analyze whether the lack of SR-BI affected
cholesterol content in embryos/fetuses.
Human Molecular Genetics, 2013, Vol. 22, No. 6 1087
RESULTS
Neural tube closure defect (NTD) and exencephaly explain
the deviation from the Mendelian ratio found in
SR-BI-deficient mice obtained from heterozygous
intercrossing
Conceptuses in SR-BI+/2 females after mating with
SR-BI+/2 males were examined at embryonic days 9.5
(E9.5), 12.5 (E12.5) and 16.5 (E16.5). At the three stages ana-
lyzed, resorptions were sporadic (0.8 + 0.2 resorptions per
female), and the total number of living embryos was normal
(8.7 + 0.3 embryos per female), when compared with those
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found in uteri from wild-type intercrosses (0.75 + 0.25 resorp-
tions and 7.8 + 0.4 embryos per female). Individual genotyp-
ing of developing embryos/fetuses showed that the proportions
of wild-type (SR-BI+/+), heterozygous and knock out indivi-
duals were similar to those expected from the Mendelian ratio
(Table 1), suggesting that SR-BI2/2 embryos underwent im-
plantation and developed at least until E16.5. However, the
morphologic analyses showed that a proportion of embryos/
fetuses at the different gestational ages exhibited neural tube
closure defects (NTDs) and exencephaly, a brain defect that
leads to perinatal death (Fig. 1). Individual genotyping of
embryos and fetuses revealed that this phenotype was almost
exclusive in SR-BI2/2 individuals (34 versus 6% in
SR-BI+/2 and 3% in SR-BI+/+ littermates) (Fig. 1 and
Table 1). Histologic analyses of brains from exencephalic
SR-BI2/2 embryos showed multiple defects consistent
with an abnormal neural tube closure. When compared with
brains from E12.5 SR-BI+/+ embryos, in which ventricles
were evident and lined by neuroepithelium (NE), brains
from E12.5 SR-BI2/2 exencephalic embryos exhibited col-
lapsed lateral ventricles with portions of NE exposed to the
amniotic fluid (Fig. 2A, B). At E16.5, brains from
SR-BI+/+ fetuses showed expanded ventricles exhibiting
two structures expected at this stage of development: transi-
tory bulges known as ganglionic eminences (GE) and cerebro-
spinal fluid-secreting choroid plexi (Fig. 2C). A cartilaginous
skull primordium and epidermis covering the brain were also
evident in these embryos (Fig. 2E). In exencephalic
SR-BI2/2 embryos, the lateral ventricles were collapsed
and the GE were not evident (Fig. 2D). Moreover, the third
and fourth ventricles were absent and coroid plexi were abnor-
mally localized, exposed to the amniotic fluid. In SR-BI2/2
exencephalic fetuses, no epidermal or cartilaginous tissue
separated the developing brain from the amniotic fluid
(Fig. 2F).
Neural tube closure involves a complex sequence of
events, including the elevation, bending and fusion of the
neural folds that require the coordinate occurrence of cell
proliferation and cell death. Immunohistochemical analyses
of cranial embryo sections at E9.5 showed that the percentage
of proliferative cells (Ki67-positive) were not different in the
neural tubes from SR-BI+/+ and SR-BI2/2 embryos with
or without NTD (data not shown). Apoptotic cells, identified
as cells showing nuclear condensation and/or blebbing and
positive staining for active caspase-3, were sporadic in
neural tubes of embryos from both genotypes (data not
shown).
1088 Human Molecular Genetics, 2013, Vol. 22, No. 6

Table 1. Number of embryos/fetuses mice from each genotype recovered from heterozygous intercrosses

Age Genotypea Total

SR-BI+/+ SR-BI+/2 SR-BI2/2
Normal NTD Normal NTD Normal NTD

E9.5 48 (96%) 2 (4%) 93 (89%) 12 (11%) 41 (70%) 18 (30%) 214

E12.5 31 (97%) 1 (3%) 61 (100%) 0 (0%) 18 (67%) 9 (33%) 120
E16.5 11 (100%) 0 (0%) 24 (100%) 0 (0%) 3 (37%) 5 (63%) 43
E(9.5+12.5+16.5) 90 (97%) 3 (3%) 178 (94%) 12 (6%) 62 (66%) 32 (34%) 377
P30 122b 202b 40b 364

P30 indicates postnatal age of 30 days.

a
Number of individuals (percentage of total).

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b 2
x P , 0.0001 versus Mendelian ratio.

Figure 1. Detection of abnormal neural tube closure and exencephaly in embryos/fetuses obtained from SR-BI+/2 intercrosses. Embryos and fetuses obtained
at E9.5 (A, D), E12.5 (B, E) and E16.5 (C, F) were photographed under a dissecting microscope. Arrows indicate an open neural tube at E9.5 (D) or exposed
brain tissue at E12.5 and E16.5 (E and F). Forceps used to keep the embryo in position are visible in (A). Bar: A, D: 500 mm; B–F: 3 mm.

Other defects in facial development in SR-BI-deficient
exencephalic fetuses
The analysis of SR-BI-deficient fetuses at E16.5 revealed add-
itional defects in facial features. First, exencephalic indivi-
duals had an open mouth with a protruding tongue, a finding
that was never observed in wild-type fetuses (Fig. 3A, B).
Histologic analysis of tongue sections from exencephalic
fetuses showed that the myogenic fibers were disorganized
in comparison to those from normal embryos, where one
central fiber was arranged in the middle of several parallel
horizontal myogenic fibers (Fig. 3C, D). In addition, only
two out of five exencephalic fetuses analyzed had completed
the fusion of the eyelids, a process that is normally completed
at E16.5 and observed in 100% of SR-BI+/+ fetuses. Histo-
logic examination of eye sections in those individuals
confirmed that the eyelids were short and never reached the
fusion area and also revealed multiple defects in the organiza-
tion of the tissues within the eye (Fig. 3E, F). As both the
neural tube and the eyelids constitute rostral structures that
require tissue extension and fusion, we analyzed palatal
shelve fusion, a developmental step involving similar pro-
cesses, in SR-BI2/2 fetuses. All fetuses analyzed had suc-
cessfully completed the fusion of the palatal shelves at
E16.5, as evidenced by the presence of a normally structured
nasopharyngeal cavity (Fig. 3G, H). Beyond the defects
described above, SR-BI2/2 exencephalic embryos were ap-
parently normal.
Non-exencephalic SR-BI2/2 embryos, corresponding to
66% of the total number of SR-BI2/2 individuals analyzed,
did not show detectable developmental abnormalities in the
brain or in other vital organs, i.e. liver and heart.
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Figure 2. Histologic confirmation of exencephaly in SR-BI2/2 abnormal fetuses. Transversal cranial sections were stained with hematoxylin and eosin. Rep-
resentative brain section from E12.5 SR-BI+/+ embryo showing lateral (LV), third (3V) and fourth (4V) ventricles lined by NE (enclosed by dotted lines) (A)
when compared with a brain section from E12.5 SR-BI2/2 exencephalic embryo exhibiting portions of neuroepithelium exposed to the amniotic fluid (indi-
cated by arrows) and collapsed lateral ventricles (B). Representative brain section from E16.5 SR-BI+/+ fetus showing expanded ventricles with evident GE
and choroid plexi and visible epidermal (E) and cartilaginous (Cg) tissue in skull primordium (C) when compared with brain from SR-BI2/2 exencephalic fetus
with collapsed lateral ventricles, absent third ventricle, open fourth ventricle and choroid plexus exposed to the amniotic fluid (D). Coronal sections from E16.5
fetuses immunostained with an antibody against the epithelial marker cytokeratin comparing the presence (E, black arrowhead) and absence (F, white arrow-
head) of epidermis surrounding the developing brain in SR-BI+/+ and SR-BI2/2 exencephalic fetuses, respectively. Bar: A, B: 1 mm. C, D: 2 mm. E, F:
200 mm.

The frequency of exencephaly is higher in SR-BI-deficient
females
As reported previously (2) and confirmed in this study, the pro-
portion of SR-BI2/2 mice weaned from our colony was lower
than expected by the Mendelian ratio. An interesting additional
observation, however, was that the proportion of 30-day-old
SR-BI2/2 females weaned was even lower than the proportion
of males and accounted for only one-third of total number of
SR-BI2/2 mice (Table 2). Previous human and mouse
studies have shown that females are more susceptible than
males to the development of NTDs (25,26). To determine if
there is an increased susceptibility of SR-BI2/2 females to
exencephaly, sex ratios were determined for NTD and normal
embryos of each genotype. Sex determination was performed
by discrimination of smcx and smcy alleles by PCR amplifica-
tion from yolk sacs or embryo tails. Results showed that
female embryos accounted for 72% of NTD SR-BI2/2 indivi-
duals, as exencephaly was significantly higher in female than in
male embryos (44% in females versus 20% in males) (Table 2),
explaining the reduced number of female mice obtained at
weaning and supporting the idea that the underlying cause of
SR-BI2/2 mice demise is indeed NTD.
SR-BI is present in different tissues of the maternal – fetal
interface
We next analyzed the localization of SR-BI in embryos and extra-
embryonic tissues from wild-type mice by immunohistochemistry
using anti-SR-BI as a primary antibody. At E9.5, SR-BI was
localized in the surface of antimesometrial trophoblast giant
cells (TGCs) that correspond to the outermost cells surround-
ing the early conceptus (Fig. 4B). Despite the association
between SR-BI deficiency and NTD, this receptor was not
detected in the embryo itself at E9.5 (Fig. 4C), suggesting
an indirect effect of TGCs on the initial differentiation of
the neural tissues. At E12.5, SR-BI was detected in the placen-
tal labyrinth, specifically in one of the two layers of syncytio-
trophoblast that separates maternal and fetal circulations. This
syncytiotrophoblast layer number III is localized between the
fetal endothelial cells and the cytokeratin-positive syncytiotro-
phoblast layer II that lines maternal sinuses (MS) (27)
(Fig. 4D, E). At E12.5, SR-BI was also present in the apical
region of visceral endoderm (VE) cells of the yolk sac
(Fig. 4F). The specificity of the antibody used for these ex-
periments was assessed by immunohistochemistry (Fig. 4B,
inset) and western blot using extraembryonic tissues from
SR-BI+/+ and SR-BI2/2 E9.5 embryos (Fig. 4G).
Despite the high expression of SR-BI in the placental laby-
rinth, no gross structural abnormalities were found by histo-
logic analysis in placentas lacking this receptor (data not
shown). Both the area of the placenta and of the labyrinth mea-
sured in sagittal sections through the midline of SR-BI+/+
and SR-BI2/2 placentas were similar (3.7 + 0.2 mm2 versus
3.3 + 0.2 mm2 and 2.1 + 0.2 mm2 versus 1.9 + 0.2 mm2 for
total area and labyrinth area from SR-BI+/+ and BI2/2 pla-
centas, respectively).
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Figure 3. Identification of defective tongue and eye structures in exencephalic SR-BI2/2 fetuses. Exencephalic SR-BI2/2 E16.5 fetuses exhibited open
mouths with protruding tongues and open eyelids (B, arrow), abnormal features that were not observed in SR-BI+/+ fetuses at this stage (A, B). Coronal sec-
tions of tongues from exencephalic SR-BI2/2 fetuses showed larger tongues with disorganized muscle fibers when compared with tongues from SR-BI+/+
fetuses, where a central vertical fiber and several horizontal, parallel myogenic fibers are observed (C, D). Histologic examination of sagittal sections indicated
that exencephalic fetuses showed abnormal eye differentiation and defective eyelid fusion (E, F). The fusion of the palatal shelves (Pal) was not affected in the
exencephalic fetuses when compared with SR-BI+/+ fetuses, as evidenced by the presence of a normal nasopharyngeal cavity (G, H). Bar: A, B: 1 mm. C–F:
200 mm. G, H: 500 mm.

Table 2. Sex proportions in SR-BI+/+ and SR-BI2/2 mice and embryos
Age Genotype Females, n (%) Males, n (%)
P30 SR-BI+/+ 63 (50%) 63 (50%)
SR-BI2/2 27a (33%) 56a (67%)
E(9.5 + 12.5) SR-BI2/2 27 (45%) 33 (55%)
SR-BI2/2 NTD 21a (72%) 8a (28%)
P30 indicates postnatal age of 30 days.
a
Fisher’s exact test, P , 0.05 versus SR-BI+/+ mice or normal SR-BI2/2
embryos.
SR-BI-deficient embryos show a moderate decrease in
their body cholesterol content
The main function of SR-BI is to mediate the uptake of esteri-
fied cholesterol and other lipids (e.g. vitamin E) from HDL
lipoproteins to cells (1,2). The relevance of SR-BI for
embryo brain development and the localization of this receptor
in cells that constitute the maternal – fetal interfaces at E9.5
and E12.5 suggest that SR-BI may be involved in the transport
of lipoprotein cholesterol across these barriers during early de-
velopment. To analyze this possibility, whole body cholesterol
content was compared in SR-BI+/+, SR-BI+/2 and
SR-BI2/2 embryos at E9.5 and E12.5. A modest, but not
statistically significant, decrease in the cholesterol content of
E9.5 SR-BI2/2 embryos when compared with their
SR-BI+/+ littermates was detected (Fig. 5A). At this stage,
cholesterol levels were highly variable among embryos in
the different genotypes. The cholesterol content of
SR-BI2/2 embryos showing NTD was similar to the one
found in SR-BI2/2 embryos without NTD. At E12.5, a sig-
nificant decrease was observed in the cholesterol content of
SR-BI deficient embryos, both SR-BI2/2 and SR-BI+/2,
when compared with their SR-BI+/+ littermates (Fig. 5B).
Again, we observed no difference in the cholesterol content
of SR-BI2/2 embryos with or without NTD.
SR-BI-deficient embryos without NTD show IUGR
It has been reported that the protein levels of SR-BI in human
term placentas are lower in pregnancies complicated by IUGR
when compared with normal pregnancies (8). To determine
the relevance of SR-BI for fetal growth in mice, we analyzed
the weight of SR-BI 2/2 embryos not exhibiting NTD at dif-
ferent stages of gestation. At E12.5, when intrauterine devel-
opment starts being almost fully sustained by a mature
placenta, SR-BI2/2 embryos were slightly lighter than
their SR-BI+/+ littermates, but this difference was not statis-
tically significant (results not shown). However, at E16.5,
SR-BI2/2 fetuses were already smaller that their littermates
(data not shown), and this defect was maintained until the day
of birth when SR-BI2/2 pups showed a small, but significant
reduction in their mean weight when compared with
SR-BI+/+ littermates (Fig. 6). Analyzing the weight of
pups individually indicated that 62% of SR-BI2/2 and
16% of SR-BI+/2 newborns were below the 10th percentile
of the weight from SR-BI+/+ littermates, demonstrating a
high prevalence of IUGR associated with the SR-BI null
genotype.
Human Molecular Genetics, 2013, Vol. 22, No. 6 1091
DISCUSSION
Previous studies reporting abnormally low numbers of
SR-BI2/2 mice weaned from SR-BI+/2 intercrosses sug-
gested the involvement of SR-BI in intrauterine development
(2). The main findings of our study were as follows: (i) a pro-
portion of SR-BI deficient embryos exhibit exencephaly, a
neural tube defect that leads to perinatal lethality, with more
prevalence in females, (ii) SR-BI is localized in specific cell
types involved in materno-fetal nutrient transport, (iii) the
lack of SR-BI results in a modest reduction in embryonic chol-
esterol levels and (iv) SR-BI 2/2 newborns with normal
brain development are smaller than their littermates.
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The existence of more than 200 mutations in different genes
leading to exencephaly in mice illustrates the complexity of
the genetic basis underlying this abnormal condition (28).
As in most mouse models of NTD (28), SR-BI2/2
embryos showed a defect specifically at the cranial region,
as spina bifida aperta was never observed in these embryos.
Two additional developmental abnormalities were detected
in rostral structures from exencephalic SR-BI2/2 embryos:
abnormal tongue morphogenesis and defective eyelid closure
associated with disorganization of eye structures, suggesting
that the differentiation of the neural tube, the eyes and the
tongue might involve a common pathogenic pathway con-
trolled by SR-BI. Other NTD mouse models with defective
eye development have been identified, although the connec-
tion between neural tube closure and eye differentiation is
not clear (28,29). Interestingly, some common morphogenetic
pathways, i.e. BMP signaling, have been found in neural tube
closure as well as eye and tongue development (30– 33).
The human condition analogous to mouse exencephaly is
anencephaly, characterized by the lack of the skull vault and
brain tissue at birth because of the degeneration of the devel-
oping brain in utero induced by the exposure to the amniotic
fluid. Both epidemiologic and experimental studies in
humans and mice have found that anecephaly and exencephaly
are more prevalent in female individuals (26). Sexing of
SR-BI2/2 developing embryos showed that the proportion
of exencephalic females doubled the proportion of males
with this defect. Although the mechanism explaining the
high prevalence of NTD in females is unknown, the current
hypothesis suggests that male and female embryos differ in
some specific aspect(s) of the neurulation process that
increases the susceptibility of females to the development of
exencephaly (25). It has been suggested that this may be
related to the high levels of methylation required to inactivate
one of the X-chromosomes (28), although this hypothesis has
not yet been fully validated.
In most of the genetic mouse models showing NTD, the
mechanism of failure of neural tube closure is attributed to
the absence of that gene function either in the neural folds
per se or in the adjacent embryonic tissues (28). The scenario
seems to be different for SR-BI, as the existing evidence
shows that this receptor is not expressed in the embryo itself
before E14.5 (our results and 6). Alternatively, the pattern of
SR-BI expression in cells from extraembryonic tissues of the
interface between mother and embryo/fetus at different
stages of development suggests the involvement of this recep-
tor in nutrient materno-fetal transport. In early gestation,
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Human Molecular Genetics, 2013, Vol. 22, No. 6
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Figure 6. Reduced weight in SR-BI2/2 pups at birth. Weights from pups
obtained from heterozygous intercrosses were expressed as the relative pup
weight (percentage of the mean weight determined in SR-BI+/+ pups from
the same litter). Different lettering above each group indicates statistically sig-
nificant differences (ANOVA; P,0.001). The dotted line indicates the 10th
percentile of SR-BI+/+ pups. ∗∗ P , 0.01 Fisher’s exact test versus
SR-BI+/+. n: SR-BI+/+ ¼ 20; SR-BI+/2 ¼ 31; SR-BI2/2 ¼ 13.

embryos before E10 (35), so other materno-fetal cholesterol

Figure 5. Moderate decrease in body cholesterol content in SR-BI2/2
embryos. The determination of total cholesterol in embryos showed that at
E9.5, the cholesterol content in embryos from each genotype was not signifi-
cantly different (A), whereas at E12.5, the SR-BI-deficient embryos contained
less cholesterol than their SR-BI+/+ littermates (B). Different lettering indi-
cates statistically significant differences (P , 0.05).
SR-BI is localized exclusively in parietal TGCs, cells with im-
portant endocrine and paracrine functions, secreting different
hormones as well as numerous angiogenesis- and hematopoi-
esis-related factors (34). Their function is to facilitate implant-
ation, to initiate decidual differentiation and maternal
vascularization of the placenta and to avoid embryo rejection.
Our data showing that the percentage of resorptions and the
total number of SR-BI2/2 embryos obtained from SR-
BI+/2 intercrosses are normal suggest that implantation or
initial placentogenesis is not affected by the lack of SR-BI.
Interestingly, cholesterol synthesis is insignificant in mouse
transport mechanisms are probably compensating for the
supply of this lipid in SR-BI2/2 embryos during neural
tube closure. On the other hand, our results linking NTD to
the lack of SR-BI in TGCs during neurulation offer a new per-
spective on the role of this cell type during early gestation.
During early development, visceral endoderm cells of the
yolk sac are functional and express lipoprotein endocytic
receptors, such as cubilin and megalin, that actively take up
lipids, proteins, ions and vitamins (19,36). Nutrient transport
across the yolk sac is important during neurulation, as
several mouse mutants deficient in proteins expressed specific-
ally in the VE exhibit NTD (18). Unexpectedly, we and others
have not been able to detect SR-BI in the yolk sac before
E10.5, suggesting that SR-BI might have a specific function
in TGCs and not in the yolk sac during neurulation. The fact
that SR-BI2/2 embryos show a reduction in whole embryo
cholesterol content suggests that TGCs might be involved in
the regulation of cholesterol uptake into the embryo either dir-
ectly or by exerting a paracrine or endocrine effect on other
tissues, i.e. the yolk sac. After E10.5, SR-BI shows a polarized
localization on the apical region of visceral endoderm cells
facing the maternal side of the conceptus (our results and 6).
Hatzopoulos et al. (6) had previously reported that SR-BI
was present in syncytiotrophoblast cells lining MS. Although
our studies also localized SR-BI to syncytiotrophoblasts, SR-
BI was detected specifically in syncytiotrophoblast layer III,

Figure 4. Localization of SR-BI in extraembryonic tissues. In (A), schematic representations of the organization of extraembryonic tissues at E9.5 and E12.5 are
shown. Upper panel, E9.5 tissues: TGCs, parietal endoderm (PE), yolk sac (YS) and amnion. Lower left panel, layers of the E12.5 placenta: decidua (Dec),
spongiotrophoblast (SpT), labyrinth (Lab). Lower right panel, details of trophoblast layers (I, II and III) at the interface of the maternal and fetal circulations
in the labyrinth at E12.5. Sections from E9.5 conceptuses immunostained with anti-SR-BI antiserum showed specific staining for SR-BI in antimesometrial TGCs
(negative control in inset) (B) and the absence of staining in the embryo proper (NE, neuroepithelium; Am, amnion) (C). In E12.5 placentas, staining for SR-BI
was observed in syncytiotrophoblast layer III, adjacent to fetal vessels (endothelial cell nuclei is indicated by an arrowhead) containing immature red blood cells
(asterisks) (D), whereas cytokeratin staining was detected in syncytiotrophoblast layer II, localized surrounding MS containing mature red blood cells (arrows)
(E). Specific staining for SR-BI was also observed in the VE of the yolk sac at E12.5 (F). Western blot analysis showed a specific band for SR-BI in SR-BI+/+,
but not 2/2 E9.5 extraembryonic tissues (1-COP was used as a loading control) (G). Bar: B, F: 25 mm. C: 100 mm. D, E: 10 mm.
1094 Human Molecular Genetics, 2013, Vol. 22, No. 6
lining fetal vessels and separated from MS by cytokeratin-
positive syncytiotrophoblasts layer II (27). Our results
support the idea that, despite their structural similarity, the
two layers of syncytiotrophoblasts separating maternal and
fetal circulations could have different molecular features prob-
ably underlying distinct functions.
An interesting observation from our localization studies is
that whereas in some cell types, such as the visceral endoderm
of the yolk sac, SR-BI exhibits a polarized cell surface local-
ization, in other cells such as TGC and syncytiotrophobasts,
this receptor does not seem to be polarized. It has been
shown that in polarized cells, SR-BI resides in caveolae and
mediates cholesterol transport across the cells via transcytosis
regulated by the cholesterol content of the cells (37,38).
Regardless of its subcellular localization, the presence of
SR-BI in one of the three layers of trophoblasts in the placenta
is in agreement with its function as a materno-fetal transporter.
Given the importance of cholesterol for brain development,
and the well-known function of SR-BI in the uptake of this
lipid in placental cells, we hypothesized that SR-BI-deficient
embryos could have reduced cholesterol levels. Surprisingly,
the reduction found in the whole body cholesterol content
was significant but modest, and there was no difference
between exencephalic and non-exencephalic SR-BI2/2 indi-
viduals. Besides its role as a cholesterol transporter, SR-BI has
also been shown to be involved in the cellular uptake of lipo-
soluble vitamins (i.e. vitamin E and vitamin D) in the intestine
and other tissues (39– 41). Thus, it cannot be ruled out that the
lack of SR-BI also affects the uptake of vitamins in extraem-
bryonic tissues that are required for neural tube closure. For
this reason, further studies will involve feeding pregnant
SR-BI+/2 females with diets supplemented with vitamin E
or other antioxidants and analyzing the prevention of NTD
in SR-BI2/2 embryos.
One of the most striking functions of cholesterol is the
modulation of the activity of Sonic hedgehog (Shh), a morpho-
gen involved in neural tube closure, among many develop-
mental processes (42– 45). An interesting hypothesis to
explain NTD in SR-BI2/2 embryos is that the decrease in
cholesterol levels may negatively affect Shh activation. In a
preliminary study, we have not found differences in the
mRNA levels of five genes regulated by Shh (Gli1, Gli2,
Patched1, Pax6 and Nkx2.1) using quantitative RT-PCR in
SR-BI+/+ versus SR-BI2/2 embryos, suggesting that Shh
signaling during development is not highly affected by the
lack of SR-BI. The transport of several morphogens in lipopro-
teins has been shown to be crucial to establish their concentra-
tion gradients (46– 48). Thus, other signaling pathways might
be dysregulated in embryos lacking SR-BI because of deficient
morphogen transport by abnormal lipoproteins.
Studies in humans have found reduced levels of SR-BI protein
in term placentas from newborns showing IUGR (8). Interestingly,
a significant proportion of SR-BI2/2 newborn mice that undergo
normal neural tube closure exhibit lower birth weights than their
wild-type littermates. Although the difference in weight detected
in SR-BI2/2 pups might seem small, it has been shown in
humans that a weight at birth below the 10th percentile seriously
affects the health of newborns and increases the prevalence of
disease in adulthood (49). In fact, two-thirds of SR-BI2/2 indi-
viduals were below the 10th percentile, indicating that in fetuses
not exhibiting NTD, the lack of SR-BI is associated with a high
prevalence of IUGR. As IUGR is associated with high perinatal
mortality and morbidity (50), this phenotype might also contribute
to the low number of weaned SR-BI2/2 embryos. Given the im-
portance of the placenta for the transport of nutrients between the
mother and the fetus, our results offer an attractive model to study
the impact of placental lipoprotein metabolism in fetal growth
(49). These results are also interesting from a clinical perspective,
as IUGR children are prone to cardiovascular disease, obesity and
type 2 diabetes in adulthood (49). The design of mouse models
lacking SR-BI specifically in the embryo and not in extraembryo-
nic tissues could be useful to understand the role of SR-BI during
fetal growth and the impact of this receptor in early life on adult
Downloaded from https://academic.oup.com/hmg/article/22/6/1086/582599 by guest on 21 December 2020
metabolism and disease conditions.
Altogether, the results of the present work suggest that the
presence of SR-BI in extraembryonic tissues is important for
the materno-fetal transport of cholesterol and/or other lipid
molecule(s) required for neural tube closure, eye and tongue
differentiation and fetal growth.
MATERIALS AND METHODS
Animals
Mice with a targeted deletion of the SR-BI gene on a mixed
C57BL/6 × 129 background (B6;129S2-Scarb1tm1Kri/J) (2)
were used in these studies. Animals were housed in a temperature-
and light-controlled room and were allowed to consume standard
chow (Prolab RMH3000, Labdiet) and water ad libitum.
Sampling of embryos and pups from heterozygous
intercrosses
Two- to three-month-old virgin SR-BI+/2 females were
housed with SR-BI+/2 males, and the presence of vaginal
plugs was checked every morning as an indication of mating.
The day when the vaginal plug was detected was recorded as em-
bryonic day 0.5 (E0.5). Protocols were conducted in agreement
with the National Research Council (NRC) publication Guide
for Care and Use of Laboratory Animals (copyright 1996, Na-
tional Academy of Science). The studies conducted were
approved by the Ethics Committee for Animal Welfare from
the School of Medicine of the Pontificia Universidad Católica
de Chile. Pregnant dams were sacrificed at 9.5, 12.5 or 16.5
days post coitum after anesthesia with a mix of ketamine:xyla-
zine (0.18 mg:0.012 mg per gram of mouse). The embryos
and extraembryonic membranes were retrieved using a dissect-
ing microscope whenever necessary, and the morphology of
each embryo was registered before fixation in Bouin’s fixative
or flash freezing in liquid nitrogen. Yolk sacs were used for
genotyping by PCR as described previously (2). Alternatively,
some dams were allowed to get to term and undergo vaginal de-
livery, and the pups were weighed and genotyped using tail tips.
Histologic and immunohistochemical analyses
Bouin-fixed tissues were dehydrated, cleared and embedded in
paraffin. Tissue sections (8 mm) were stained with hematoxylin
and eosin or used for immunohistochemical procedures. For
immunolocalization, sections were subjected to antigen

retrieval using hot citrate and then incubated with a primary
antibody against SR-BI developed in rabbit (1:200, 6) or com-
mercial rabbit antibodies against cytokeratin (1:200, Dako),
Ki67 (1:200, Abcam) or cleaved caspase 3 (1:200,
Promega). After extensive washing, sections were incubated
with secondary antibodies coupled to peroxidase (1:800,
Sigma) and revealed with diaminobenzidine (Sigma). Sections
from two tissues known to express SR-BI, ovary and liver
were used as positive controls. Negative controls included
slides where the primary antibody was replaced by non-
immune rabbit serum, and sections where SR-BI2/2 tissues
were incubated with anti-SR-BI antibody.
Sex determination of embryos by PCR
Individual sexing of embryos was performed by PCR as
described elsewhere (51). The following primers F: 5′ -CCGC
TGCCAAATTCTTTGG-3′ and R: 5′ -TGAAGCTTTTGGCT
TTGAG-3′ were used to amplify bands of different sizes cor-
responding to the smcx/y gene in the X and Y chromosomes.
Immunoblotting
Tissues from E9.5 conceptuses and E12.5 embryos were ana-
lyzed by immunoblot as described elsewhere (2).
Cholesterol determinations
Whole embryos were homogenized in lysis buffer [25 mM Tris
pH 7.5, 150 mM NaCl, 5 mM EDTA pH 8, 1% IGEPAL
(Sigma), 0.01% SDS and 0.35 mg/ml PMSF], and lipids
were extracted in methanol:chloroform 1:2 following standard
procedures. The cholesterol content in E12.5 embryos was
determined using an enzymatic colorimetric assay previously
reported (52). In E9.5 embryos, the cholesterol content was
determined using the Amplex Red Cholesterol Assay (Invitro-
gen), following the manufacturer’s instructions, because of a
higher sensitivity of this test. Briefly, extracts were dissolved
in reaction buffer and incubated with cholesterol oxidase, per-
oxidase and the Amplex Red reagent. The enzymatic oxidation
of cholesterol drives the stoichiometric oxidation of the
Amplex Red reagent to resorufin. The fluorescence of the
product was measured on a microplate fluorimeter with exci-
tation at 530 nm and emission reading at 590 nm. Results
were expressed as nanogram of cholesterol per microgram of
protein in the lysate. In each experiment, a known quantity
of pure cholesterol was processed in parallel to the samples
for correction because of extraction efficiency.
Statistical analyses
The data are presented as mean + SEM, unless stated other-
wise. The significance of the differences between means was
tested using ANOVA and Tukey’s post hoc test. To compare
proportions against an expected value, x2 analyses were per-
formed. The difference between binominal outcomes in two
groups was tested using Fisher’s exact test. Differences were
considered significant at P , 0.05. Where ANOVA with
Tukey’s post hoc test was performed, different lettering indi-
cates statistically significant differences.
Human Molecular Genetics, 2013, Vol. 22, No. 6 1095
ACKNOWLEDGEMENTS
We would like to thank Dr Monty Krieger and Dr Richard
Haynes for useful discussions. We also acknowledge Jenny
Corthorn and Ludwig Amigo for their technical assistance in
performing the histologic analyses and cholesterol determina-
tions, respectively.
Conflict of Interest statement: The authors do not have any
conflict of interest.
FUNDING
Downloaded from https://academic.oup.com/hmg/article/22/6/1086/582599 by guest on 21 December 2020
This work was supported by the Chilean National Council for
Scientific and Technological Research (CONICYT) programs
Inserción de Investigadores Jóvenes en Academia (79090028
to D.B.) and Fondo Nacional del Desarrollo Cientı́fico y Tec-
nológico (11090064 to D.B. and 1110712 to A.R.) and by the
Summer Research Program for Medical Students from the
School of Medicine, Pontificia Universidad Católica de
Chile (to I.P.).
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