430 A.

DUGGAN
JOURNALET
OFAL.
EXPERIMENTAL ZOOLOGY 292:430–434 (2002)
DOI 10.1002/jez.10067

Expression of SR-BI (Scavenger Receptor Class B

Type I) in Turtle (Chrysemys picta) Tissues and
Other Nonmammalian Vertebrates
ANNEMARIE E. DUGGAN,* RONALD STE. MARIE JR., AND
IAN P. CALLARD
Department of Biology, Boston University, Boston, Massachusetts 02215

ABSTRACT In this study, the tissue distribution of the expression of an HDL-receptor (SR-BI;
Scavenger Receptor Class B Type I) was investigated in the turtle using an antiserum to murine
SR-BI. Several turtle tissues including liver, heart, small intestine, kidney, oviduct, ovary, and
testis were shown to express an 82 kDa membrane protein. In addition, SR-BI expression in livers
of other nonmammalian species such as the chicken, frog, goldfish, shark, and skate is also re-
ported. Ovarian SR-BI expression varies seasonally in the turtle as do plasma levels of apoA-I and
cholesterol ester. It is possible that changing levels of SR-BI, the receptor for apoA-I, is physiologi-
cally relevant to the demands of the turtle ovarian cycle and cholesterol distribution. J. Exp. Zool.
292:430–434, 2002. © 2002 Wiley-Liss, Inc.

Cholesterol and cholesterol esters are hydro-
phobic lipids that are transported in the aque-
ous environment of the bloodstream in the form
of lipoproteins. Cholesterol and its esters are de-
livered to cells in the form of LDL (low density
lipoprotein) through recognition of apoB (the pro-
tein component of LDL) by the LDL-Receptor
(Gotto et al., ’86). This uptake of the LDL par-
ticle occurs via receptor-mediated endocytosis
(Brown and Goldstein, ’86). In contrast to LDL,
HDL (high density lipoprotein) takes up free cho-
lesterol from peripheral tissues where it then
becomes esterified within the bloodstream. This
cholesterol ester of HDL is then delivered pri-
marily to the liver and steroidogenic tissues
(Steinberg, ’96). The recycling of circulating cho-
lesterol back to the liver for degradation and fur-
ther elimination is known as reverse cholesterol
transport (Fielding and Fielding, ’95). The pri-
mary ligand for HDL is apoA-I, which is thought
to interact transiently with a different receptor,
SR-BI (scavenger receptor class B Type I) in a
nonendocytotic process. This receptor selectively
takes up cholesterol ester from HDL particles
without endocytosis of the particle in murine
LDL-receptor-negative CHO cells (Acton et al,
’96). Because SR-BI is preferentially expressed
in liver and steroidogenic cells, it plays an im-
portant direct role in cholesterol homeostasis, as
well as indirectly through its availability as a
steroid precursor.
The present study was initiated to determine
whether SR-BI is present in nonmammalian spe-
cies, the chicken (Gallus domesticus), the fresh-
water turtle (Chrysemys picta), an amphibian
(Xenopus laevis), a teleost fish, the goldfish (Car-
assius auratus), and the elasmobranchs (Squalus
acanthias and Raja erinacea). Here we report the
identification of SR-BI receptor by Western analy-
sis using an antibody to mouse SR-BI. The
receptor is highly expressed in liver, ovary, inter-
renal, heart, and blood vessels in the turtle and
in the liver in the chicken, frog, goldfish, shark,
and skate. These results suggest that the SR-BI
receptor is phylogenetically ancient and is struc-
turally conserved as indicated by the strong cross-
reactivity of fish, amphibian, reptilian, and avian
tissues with mammalian antibody.
MATERIALS AND METHODS
Animals
Turtles were obtained from Lemberger Co.
(Oshkosh, WI) and maintained in aquarium tanks
until autopsy. Rat liver tissue was provided by
D. Waxman, and goldfish and shark liver tissues
were provided by M. Betka. Chicken liver tissue
Grant sponsor: NIHRR 06633 to IPC.
*Correspondence to: Annemarie Duggan, New England Regional
Primate Research Center, One Pine Hill Drive, Harvard University,
Southborough, Massachusetts 01772.
E-mail: annemarie_duggan@hms.harvard.edu
Received 17 May 2001; Accepted 8 November 2001

© 2002 WILEY-LISS, INC.

 SR-BI EXPRESSION IN TURTLE TISSUES
was provided by T. Gilmore and frog liver tissue
by the Boston University Laboratory Animal Care
Facility. Skate liver, ovary, and epigonal organ
were obtained by I.P. Callard at Mount Desert
Island Biological Laboratory.
Membrane preparations for tissues
All tissues were homogenized in buffer (20 mM
Tris-HCl, 1 mM CaCl2, 111 mM NaCl, 1 mM
PMSF, 2 uM leupeptin) and spun at 5,000 rpm
for 10 mins at 4°C. Supernatants were then spun
at 33,000 rpm for 45 mins at 4°C. The resultant
pellets were resuspended in Laemmli buffer us-
ing a 22.5-gauge needle.
Protein determination and SDS-PAGE
Protein concentrations of extracts were deter-
mined using the Lowry method. Fifty micrograms
(50 µg) of protein from tissue extracts were re-
duced using beta-mercapto-ethanol and were run
on 7.5% SDS-polyacrylamide gels. Prestained
markers from Bio-Rad (Hercules, CA) were used
as reference standards.
Western blot analysis
Proteins were transferred from the gels to ni-
trocellulose membranes at 20 V for 1 hr using a
semi-dry transfer cell (Bio-Rad). Membranes
were incubated overnight in 5% milk in TBS
buffer. They were then washed in Tween-20-TBS
3x for 5 mins followed by incubation for 2 hrs
with primary antibody (anti-mouse SR-BI; pro-
vided by Millennium Pharmaceuticals, Cam-
bridge, MA) in 5% milk/Tween-20/TBS at a
dilution of 1:500, 1:2000. The membranes were
washed again in the same manner and incubated
for 1 hr with secondary antibody (anti-rabbit IgG
conjugated to alkaline phosphatase; Sigma-
Aldrich, St. Louis, MO) under the same condi-
Fig. 1. SR-BI cross-reactivity in mouse tissues. Western
blot analysis of mouse tissue membrane extracts using anti-
mouse SR-BI. Fifty micrograms (50 µg) of protein from tis-
431
tions. After a final wash, proteins were visual-
ized using the NBT-BCIP substrate system from
Promega (Madison, WI).
RESULTS
Western blotting
SR-BI cross-reactivity in mouse tissues
(Fig. 1)
The antibody (anti-mouse) cross-reacted most
strongly with an 82 kDa membrane protein in
liver and adrenal gland followed by testis and
heart. No cross-reactivity was detected in small
intestine, kidney, or adipose tissues.
Phylogenetic distribution of SR-BI in liver
(Fig. 2)
In bird, turtle, frog, and goldfish liver samples,
an 82 kDa band cross-reacted strongly with anti-
body; in rat and shark, the reaction was less
strong, and it was weak in the skate. Frog and
goldfish proteins were slightly higher in molecu-
lar weight (∼84–86 kDa vs. 82 kDa).
Tissue distribution and reactivity of SR-BI:
cross-reactivity in turtle (Fig. 3)
The antibody cross-reacted with a membrane
protein at 82 kDa (SR-BI) most strongly in liver,
adrenal, heart, blood vessels, and ovarian follicles.
Less cross-reactivity was detected in small intes-
tine, kidney, and oviduct. Little cross-reactivity
was seen in adipose tissue.
Ovarian follicles expressed the cross-reacting pro-
tein in a size-specific manner. Very small (< 2 mm)
and large-sized (∼15 mm) follicles expressed the pro-
tein most strongly compared to a weak reaction in
small and midsize follicles. The antibody also cross-
reacted strongly with testis membrane extracts, in-
dicating that SR-BI is expressed in male tissues.
sue extracts were loaded onto 7.5% SDS-polyacrylamide gels
under denaturing conditions as described in Materials and
Methods.
432 A. DUGGAN ET AL.
Fig. 2. Phylogenetic distribution of SR-BI in liver. West-
ern blot analysis of liver membrane extracts from represen-
tatives of other vertebrate classes using anti-mouse SR-BI.
Fifty micrograms (50 µg) of protein from liver extracts were
loaded onto 7.5% SDS-polyacrylamide gels under denaturing
conditions as described in Materials and Methods.
SR-BI cross-reactivity in turtle ovarian
follicular membranes during annual cycle
(Fig. 4)
The follicular membranes (primarily granulosa
and thecal cells) from the largest ovarian follicles
present at several stages of the ovarian cycle were
extracted for Western blotting. There are clear-
cut seasonal variations: Expression is highest in
20 mm follicles just prior to ovulation in May. Af-
ter ovulation, but before egg-laying, the remain-
ing large follicles (∼16.0 mm) are less reactive;
after oviposition (June), the 15.0 mm ovarian fol-
licles yield a very strong reaction. In the fall, this
same class of follicles (∼16.0 mm in size) reacts
only weakly.
DISCUSSION
In this study we have demonstrated cross-reac-
tivity of an 82 kDa protein in the liver of bird,
turtle, frog, teleost, shark, and skate and in most
turtle tissues tested. The cross-reactivity and
molecular weight suggest significant homology of
the nonmammalian membrane protein with the
SR-BI of the mouse. That is the first indication of
this receptor in nonmammalian tissues. Its wide
phylogenetic distribution (amphibians, reptiles,
bony fish, and cartilaginous fish) suggests it is
highly conserved and of physiological importance.
It is of interest that the molecular weight of the
putative SR-BI is quite similar in different verte-
brate groups, differing only slightly in the teleost
fish. The tissue distribution in nonmammals sug-
gests it is important in a wide array of ste-
roidogenic (ovary, interrenal) and nonsteroidogenic
(liver, heart, small intestine, kidney, and oviduct)
tissues in nonmammals compared to mammals
(liver only and steroidogenic tissues). It is pos-
sible that SR-BI emerged relatively early in ver-
tebrate evolution possessing several metabolic
functions and, subsequently, became responsible
for more specialized functions in mammals.
There have been no reports of SR-BI expression
in the arterial walls of mammals or nonmammals,
but these studies have shown that turtle blood ves-
sels express SR-BI. It may be involved here in the
removal of cholesterol esters from the circulation,
which may have potential relevance with respect
to the development of atherosclerosis if expressed
in mammalian blood vessels.
Although SR-BI is reported to have limited tis-
sue distribution in the mouse (liver and ste-
roidogenic tissues), SR-BI mRNA appears to be
expressed in other nonsteroidogenic tissues in-
cluding spleen, kidney, small intestine, adipose
tissue, skeletal muscle, heart muscle, and lung
(Johnson et al., ’98). Whether or not the SR-BI
mRNA is translated into protein in these tissues
is another issue, but the presence of its message
suggests that the receptor could be widely ex-
pressed, but to a lesser degree, in extrahepatic
and nonsteroidogenic tissues. This study has dem-
onstrated that SR-BI is expressed to the same
degree in heart tissue as in testicular tissue in
the mouse, suggesting that the tissue distribu-
tion of SR-BI may not be limited to hepatic and
steroidogenic tissues.
The seasonal variation in levels of SR-BI in ova-
rian follicular extracts in the context of circulat-
ing cholesterol ester levels is of interest. Because
SR-BI selectively takes up cholesterol ester from
the circulation in mammals, these two parameters
may be inversely related. In a recent study, Varban
et al. (’98) showed a 50%–70% increase in total
plasma cholesterol accompanied by a 53% decrease
in SR-BI expression. A similar inverse relationship
appears to hold true when comparing circulating
cholesterol ester levels to SR-BI expression in the
turtle. Thus, when ovarian SR-BI is high in the
pre-ovulatory period cholesterol ester levels are
lower (123 ± 23 mg/dL; Duggan et al., 2001), sug-
 SR-BI EXPRESSION IN TURTLE TISSUES
Fig. 3. Tissue distribution and reactivity of SR-BI: cross-
reactivity in turtle. Western blot analysis of turtle tissue mem-
brane extracts using anti-mouse SR-BI. Fifty micrograms (50
µg) of protein from tissue extracts were loaded onto 7.5% SDS-
polyacrylamide gels under denaturing conditions as described
in Materials and methods. (A) indicates SR-BI cross-reactiv-
ity in several extra-gonadal turtle tissues (liver, adrenal,
heart, small intestine, kidney, oviduct, adipose, and blood ves-
sels). (B) and (C) demonstrate SR-BI cross-reactivity in sev-
eral sizes (= 15mm, 10–15 mm, 5–10 mm, and < 2 mm) of
ovarian follicles (B) in addition to testicular tissue (C; three
separate samples).
Fig. 4. SR-BI cross-reactivity in turtle ovarian follicular
membranes during the annual reproductive cycle. Western blot
analysis of ovarian follicular membrane extracts from pre-ovu-
latory, postovulatory, postoviposition, and fall stages of the re-
productive cycle using anti-mouse SR-BI. Fifty micrograms (50
µg) of protein from follicle extracts were loaded onto 7.5% SDS-
433
gesting a functional relationship between the two.
In the postovulatory period, cholesterol levels are
higher (226 ± 22 mg/dL) and SR-BI expression is
lower. After egg-laying (postoviposition), the lev-
els of cholesterol ester are declining (159 ± 23 mg/
dL) as SR-BI expression increases. Finally, in the
fall, when cholesterol ester is the highest (268 ±
10 mg/dL), SR-BI expression is the lowest. These
changes may be related to ovarian steroid levels
as in other species, where the pre-ovulatory and
follicular growth phases (spring and fall) are es-
trogen dominated, and the short luteal phase
(postovulatory) is progesterone dominated (Jones
et al., ’83). Whatever the regulatory mechanisms
may be for SR-BI, the lower SR-BI expression and
high plasma cholesterol ester in the fall suggest
that cholesterol may be diverted away from the
ovary. Possibly, cholesterol ester levels and SR-BI
expression are coupled so as to aid in the supply
of appropriate amounts of cholesterol substrate to
the ovary for steroidogenesis or as a nutrient sup-
ply for oogenesis. The expression of SR-BI in the
testis further suggests that it is involved in pro-
viding substrate for steroidogenesis. In preliminary
work, we have shown that turtle HDL can sup-
port testicular steroidogenesis in turtle testicular
incubates (Duggan, unpublished).
Hepatic expression of SR-BI in the turtle and
the other species studied suggests that a reverse
cholesterol transport pathway may be universal
in vertebrates. Once redistributed to the liver, cho-
lesterol is eliminated, in part, via bile acid me-
tabolism. The SR-BI overexpression studies in
polyacrylamide gels under denaturing conditions as described
in Materials and Methods. Follicular sizes and plasma choles-
terol ester values for months/stages shown are indicated for
comparison. Cholesterol ester values were obtained by the labo-
ratory of D. Small (BUSM, Dept. of Biophysics) using the
method of Allain et al. (’74) and Warnick (’86).
434 A. DUGGAN ET AL.
mice (Varban et al., ’98) demonstrate that biliary
cholesterol is increased, supporting this conten-
tion. Turtles have very large gall bladders (bile
volume is approximately 3–5 mls; personal obser-
vation) and it is likely that future investigation
of bile acid production in turtles and other non-
mammalian species will reveal seasonal variations
in concert with cholesterol mobilization for ova-
rian growth. In addition, apoA-I, the ligand for
SR-BI, appears to be present in plasma of some
nonmammals (turtle, chicken, and frog) tested as
well (Hermier, ’85; Paolucci and Callard, ’95;
Paolucci et al., ’98).
In summary, this is the first time that a puta-
tive HDL-receptor (mouse SR-BI homolog), impor-
tant in the selective uptake of HDL cholesterol
esters, has been reported in nonmammalian ver-
tebrates. SR-BI is widely distributed in many tis-
sues and may function to supply cholesterol for
steroidogenesis as well as for other general meta-
bolic and structural purposes. It is likely that SR-
BI is structurally and functionally conserved
phylogenetically. Further studies must be con-
ducted to ascertain the functional role of SR-BI in
nonmammalian (i.e., turtle) tissues to determine
if this is a physiologically relevant HDL-receptor.
In future work, analysis of SR-BI cDNA in tissues
of these species should be done to elucidate se-
quence similarities to mammalian SR-BI cDNA.
ACKNOWLEDGEMENTS
Thanks are due to Millenium Pharmaceuticals
for their generous donation of SR-BI antibody.
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