A specific IGF-2 receptor antiserum was used to reveal the presence of IGF-2 receptors during preimplantation development of mice. Receptors were present on 2-, 4- and 8-cell embryos, morulae, blastocysts, and on ICMs isolated prior to staining. There was no evidence for receptors on fertilized eggs. These observations confirm reports of the expression of IGF-2 receptor mRNA as early as the 2-cell stage and refine similar observations in blastocysts to confirm expression in both the TE and ICM. A potential auto/paracrine loop is thus one of the first products of activation of the embryonic genome and is expressed constitutively through preimplantation development

Harvey M.B.

Kaye P.L.

University of Queensland eSpace

Development 111, 1057-1060 (1991)
Printed in Great Britain © The Company of Biologists Limited 1991
1057
IGF-2 receptors are first expressed at the 2-cell stage of mouse
development
MARK B. HARVEY and PETER L. KAYE*
Department of Physiology and Pharmacology, The University of Queensland, Australia 4072
* Author for correspondence
Summary
A specific IGF-2 receptor antiserum was used to reveal
the presence of IGF-2 receptors during preimplantation
development of mice. Receptors were present on 2-, 4-
and 8-cell embryos, morulae, blastocysts, and on ICMs
isolated prior to staining. There was no evidence for
receptors on fertilized eggs. These observations confirm
reports of the expression of IGF-2 receptor mRNA as
early as the 2-cell stage and refine similar observations in
blastocysts to confirm expression in both the TE and
ICM. A potential auto/paracrine loop is thus one of the
first products of activation of the embryonic genome and
is expressed constitutively through preimplantation
development.
Key words: IGF-2, receptor, preimplantation embryo,
immunohistochemistry.
Introduction
Insulin and insulin-like growth factors -1 and -2 (IGF-1,
-2) promote metabolism and growth in many cell types
(Froesch and Zapf, 1985). In the preimplantation
mouse embryo, both insulin (Gardner and Kaye, 1991;
Harvey and Kaye, 1990a) and IGF-1 (Harvey and Kaye,
1989) stimulate cell division and blastocyst formation in
vitro via their own receptors.
However, IGF-2 has been suggested as the specific
embryonic/fetal member of this growth factor family
(Moses et al. 1980). Although displacement of insulin
from receptors by a mixture of IGFs-1 and -2 (Mattson
et al. 1988) and demonstration of IGF-2 receptor
mRNA in compacted embryos have been reported, the
actual time of first expression of this mRNA has been
placed between the 2-cell (Rappolee et al. 1989a; Werb,
1990) and 8-cell stages (Heyner et al. 1989; Rappolee et
al. 1989/?). On the other hand, IGF-2 mRNA has been
revealed in 2-cell embryos (Heyner et al. 1989).
In an attempt to define the ontogeny of the IGF-2
receptor (or type 2 mannose-6-phosphate receptor) and
thus the potential for auto/paracrine regulation at this
early developmental stage, a specific IGF-2 receptor
antibody (Scott and Baxter, 1987) has been used to
define the expression of the receptor in embryonic cell
membranes immunohistochemically.
Materials and methods
Superovulation and Embryo Manipulation
Randomly bred, Quackenbush mice (10 weeks) were super-
ovulated by i.p. injection of lOi.u. PMSG at 10.00 h followed
48h later by lOi.u. hCG (Folligon and Chorulon, Intervet,
Aust.) and paired with males. Mating was determined by the
presence of a vaginal plug by 9.00 h the following morning.
Immunohistochemistry
Fertilized eggs, 2-, 4- and 8-cell embryos, and blastocysts were
collected 24 h, 48 h, 60 h, 72 h, and % h post-hCG respectively
in M2 (Fulton and Whittingham, 1978; modified as previously
described, Hobbs and Kaye, 1985). IGF-2 receptors were
visualized by modification of the procedure used for insulin
receptors (Harvey and Kaye, 1991). Briefly, the zona
pellucidae were removed by brief exposure to acid Tyrode's
solution (pH 2.5; Hogan et al. 1986), before fixation with 2 %
paraformaldehyde in 10 mM phosphate buffer (pH7.2) and
transferred to 1:600 dilution of rabbit anti-rat IGF-2 receptor
IgG (Scott and Baxter, 1987) or control rabbit IgG (1:600
dilution). After 90min, embryos were washed and the bound
IGF-2 receptor IgG 'tagged' with a biotinylated, donkey anti-
rabbit IgG (1:200 dilution; Amersham Aust. Pty Ltd, Nth
Ryde, NSW, Aust.) and streptavidin-linked Texas red
complex (1:100 dilution; Amersham Aust.). Embryos were
then mounted in glycerol and visualized under a photomicro-
scope.
ICM Isolation
ICMs were isolated from blastocysts by immunosurgery
(Solter and Knowles, 1975; Harvey and Kaye, 1991).
Results
Blastocysts that are reported to express IGF-2 receptor
mRNA (Rappolee et al. 1989a,b) and from which
1058 M. B. Harvey and P. L. Kaye
1A 2A
4A
5A
9A 1OA
',-: ,
(' " \
Figs 1-10. Fixed and stained mouse preimplantation embryos (A) light microscopy (B) fluorescence microscopy; (Fig. 1
and 9) blastocyst (x250) and isolated ICM (x400), respectively, incubated with control IgG (Fig. 2) blastocyst incubated
with 130/iM IGF-2 prior and during IGF-2 receptor IgG incubation (Fig. 3-8, 10) fertilized egg, 2-, 4- and 8-ceLl embryo,
morulae, blastocyst (x250) and isolated ICM (x400), respectively, incubated with IGF-2 receptor IgG.
IGF-2 receptors on mouse embryos 1059
insulin can be displaced by IGF-2 (Mattson et al. 1988)
and whose metabolism was stimulated by IGF-2
(Rappolee et al. 1989a) were used as controls to confirm
the procedure.
No staining was present on blastocysts or isolated
ICMs incubated with the control rabbit IgG (Figs 1, 9)
or on fertilized eggs (Fig. 3). IGF-2 receptors were
visible on 2-, 4- and 8-cell embryos, morulae, blasto-
cysts and isolated ICMs (Figs 4,5,6,7,8,10). Consistent
staining was seen on embryos at these stages from at
least 3 experiments. In the majority of early cleavage
stage embryos observed, there was a greater degree of
staining on approximately 50% of the blastomeres
present (Figs 4,5,6). The specificity of this staining to
IGF-2 receptors was demonstrated by the absence of
staining on blastocysts when the primary antibody
incubation was omitted (data not shown) or a marked
decrease in fluorescence on blastocysts incubated with
130/XM IGF-2 (LiDy Research Laboratories, Indianapo-
lis, Indiana USA) prior to fixation, and before and
during IGF-2 receptor IgG incubation (Fig. 2).
Discussion
The aim of this study was to investigate if, and when,
IGF-2 receptors appeared on embryonic cell mem-
branes during preimplantation development and on
which cell population of the blastocyst they were
expressed. The specificity of the receptor antibody for
the IGF-2 receptor used in this study has been
established by the complete abolition of specific
staining on hepatocytes and hepatoma cells following
preincubation of the antiserum with purified rat type 2
receptor and by its reaction with a single species of
relative molecular mass 210000-220000 (consistent
with that of the IGF-2 receptor) when liver and
hepatoma cell micosomal membranes were western
blotted after electrophoresis (Hartshorn et al. 1989).
This antibody has also been used to show specific IGF-2
receptor staining on developing mouse sceral cells
(Cuthbertson et al. 1989).
Staining was present on embryos from the 2-cell stage
through to blastocysts (Figs 4-8), correlating with the
presence of IGF-2 receptor mRNA in 2-cell and later
stage embryos detected using reverse transcription and
polymerase chain reaction (Rappolee et al. 1989a,b).
As there is no other evidence for the presence of insulin
or IGF-1 receptors prior to the 8-cell stage (Rappolee et
al. 1989a,6; Harvey and Kaye, 1988, 1990, 1991) and
these ligands may cross react with IGF-2 receptors
(Froesch and Zapf, 1985), the early appearance of IGF-
2 receptors at the 2-cell stage may explain the early
growth effects of insulin (Gardner and Kaye, 1991) and
IGF-1 (Harvey and Kaye, 1989).
Tight junctions between cells of the TE prevent
access of antibodies to the ICM (Harvey and Kaye,
1991), so the staining on compacted embryos reveals
the presence of IGF-2 receptors only on the outer TE
cells. Immunosurgical isolation of ICMs prior to
fixation and staining (Fig. 10) confirmed the presence of
IGF-2 receptors on this cell population. It is thus
apparent that IGF-2 receptors are present from the
later 2-cell stage until the first differentiation which
occurs at compaction (at the 8-cell stage) and are then
expressed in both newly differentiated cell populations.
This ontogeny of the IGF-2 receptor supports the
suggestion that it may be a product of the embryonic
genome (Werb, 1990) that is activated in the 2-cell stage
(Flach et al. 1982) as receptors were not evident on
fertilized eggs (Fig. 3). As such, it is the first receptor in
the insulin family to be expressed during development,
and its confirmed expression appears to be constitutive
in both cell populations of the blastocyst. Since
production of IGF-2 mRNA has been observed in
embryos of the same stages, concurrent appearance of
receptor and ligand suggest auto/paracrine actions by
IGF-2 on embryonic development from the time of
activation of the embryonic genome. This contrasts
with insulin, which does not appear to be expressed by
embryos at any preimplantation stage (Rappolee et al.
1989a) and whose receptor is not expressed until
compaction of 8-cell embryos begins (Rappolee et al.
1989a,b; Harvey and Kaye, 1988, 1990).
It will be of interest to compare actions of IGF-2 on
embryonic metabolism and development with those of
the other members of the insulin family, since insulin
receptors appear to regulate mitotic and metabolic
effects whilst IGF-1 receptors only mediate mitosis
(Harvey and Kaye, 1989).
We thank Dr R. Baxter (Royal Prince Alfred Hospital,
Sydney, Aust.) for the generous gift of IGF-2 receptor
antibody and Thomas L. Jeatran, of Lilly Research Labora-
tories, Indianapolis, Indiana USA for the IGF-2, Lindsay
Shannon for photography, and Kathryn Markham for
technical assistance. This work was supported by a Research
Project Grant to P.L.K. and a Biomedical Postgraduate
Research Scholarship to M.B.H. from the National Health
and Medical Research Council of Australia.
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{Accepted 3 January 1991)


IGF-2 receptors are first expressed at the 2-cell stage of mouse development

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IGF-2 receptors are first expressed at the 2-cell stage of mouse development

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