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Development 108, 491-495 (1990)
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Insulin and insulin-like growth factor I (IGF I) in early mouse embryogenesis
RAD AN SPAVENTI, MARIASTEFANIA ANTICA and KRE$IMIR PAVELIC
Ruder BoSkovid Institute, 41001 Zagreb, Bijenitka 54, P.O. Box 1016, Yugoslavia
Summary
Growth factors have an important role in the regulation
of cell growth, division and differentiation. They are also
involved in the regulation of embryonic growth and
differentiation. Insulin and insulin-like growth factor I
(IGF I) play an important part in these events in the later
stages of embryogenesis, when organogenesis is completed. In this study, we are presenting evidence that
insulin and IGF I are also secreted by embryonic tissues
during the prepancreatic stage of mouse development.
We found measurable amounts of insulin and IGF I in 8to 12-day-old mouse embryos. We also showed that
embryonic cells derived from 8-, 9- and 10-day-old
mouse embryos secrete insulin, IGF I and/or related
molecules. Furthermore, the same growth factors, when
added to the culture of 9-day-old mouse embryonic cells,
stimulate their proliferation. These results lead to the
conclusion that insulin can stimulate the growth of
embryonic cells during the period when pancreas is not
yet formed, which is indirect evidence for a paracrine (or
autocrine) type of action.
Introduction
The aim of this study was to investigate the role of
insulin and IGF I at early stages of mouse embryogenesis and to find out whether these peptides are detectable in the 8- to 12-day-old embryos. We also tested the
effects of insulin and IGF I on primary cultures of
mouse embryonic cells, as well as the capability of these
cells to secrete insulin and IGF I.
Growth control can be mediated directly either by
insulin, which can bind to the specific receptors and IGF
receptors type I (Hill and Milner, 1985), or by the
locally secreted IGF I (autocrine and paracrine growth
control) (D'Ercole et al. 1980). Indirect regulation is
mediated by insulin (Brinsmead and Liggins, 1979;
Philips and Vassilopoulou-Sellin, 1980; Binoux et al.
1980, 1982) and placental lactogen (Hill et al. 1979)
stimulation of IGF I secretion from the liver (and
possibly kidney). Insulin and insulin-like growth factor
I (IGF I) can also stimulate growth and differentiation
of embryonic tissues grown in culture, depending upon
cell types and developmental stage (Ridpath et al. 1984;
Puro and Agardh, 1984; Ewton and Florini, 1981;
Pratten et al. 1988; Cooke and Nicoll, 1984; DePablo
et al. 1985). Travers et al. (1989) showed that explanted
rat embryos require insulin for normal development
between 9.5 and 11.5 days. Also, Sadler et al. (1986)
presented data about somatomedin inhibitors from
diabetic rat serum that alter growth and development of
9- to 10-day-old mouse embryos in culture. These in
vitro studies suggest a possible role of insulin-like
molecules as growth mediators during development. If
this is so, the question is whether these peptides
mediate fetal growth during the whole gestation period
or just during the second half of it, when all prerequisites (growth hormone secretion, mature /3 cells, developed circulatory system, etc.) exist. Although there is
some indirect evidence that insulin and IGF I could act
at a time when the organogenesis is not completed, this
still remains a matter of question.
Key words: insulin, IGF I, mouse embryogenesis.
Materials and methods
Embryos
Inbred mice 4-6 weeks old of the CBA/H Zgr strain were
obtained from pedigree lines maintained at the Ruder BoSkovid Institute (Zagreb, Yugoslavia) animal facility. Fetal mice
were obtained from timed matings as previously described.
The day of finding a vaginal plug was designated as 1st day of
embryonic development (Sadler, 1979). 8- to 12-day-old
mouse embryos were used. On the prescribed day, embryos
were removed from the uterus and placed on a watch glass
containing sterile cold PBS. Under a dissecting microscope,
embryos were separated from extraembryonic tissues including the yolk sac.
Homogenates of embryonic tissue
Whole embryos (from at least 3 mice) were homogenized in
an all-glass Potter-Elvehjem homogenizer. The homogenates
were dissolved in 0.1 M-PBS and centrifuged at 70 000 g for 1 h
at 4°C. The supernatants were used for insulin, C-peptide and
IGF I determination. All experiments were done in triplicate.
In some experiments, uterus homogenates of the same mice
were used as controls.
Serum-free medium
This was prepared by supplementing pure Dulbecco-modified
492
R. Spaventi, M. Antica and K. Pavelid
Eagle's medium (DMEM) with EGF (50ngmr'; Collaborative Research) and transferrin (25/igmr 1 , Sigma).
Primary cultures of embryonic cells
These were prepared according to the method of Todaro and
Green (1963). After fine mincing of the whole embryos, they
werefinallydissaggregated with 0.25 % trypsin (Serva). After
three subsequent washings in the cold with Hank's solution
the cells were resuspended in DMEM containing 10% FCS
and incubated for l h at 37CC. The cells were counted,
adjusted at a concentration of lO^ml"1 and seeded in either
24-well plates at afinalconcentration of 105 cells per well or in
60 mm Petri dishes at SxlO^cells per dish.
After cultivating the cells in the 24-well plates for 3 days in
DMEM with 10% FCS, we replaced it with serum-free
medium with the addition of insulin (10id.u. ml"1; Novo
Industrii) or IGF I (10 ng ml"'; AmGen). In the control wells,
only serum-free medium was added. Three days later the
number and viability of cells was determined by trypan blue
exclusion test. The results concern only the viable cells.
The concentrations of insulin and IGF I were assessed in
the conditioned medium of the cells cultivated in Petri dishes.
These cultures were first incubated in DMEM with 10 % FCS
for 3 days. After washing the cultures three times with PBS,
the serum-free medium was added and left for the next 3 days.
The amount of growth factor in serum-free medium was
measured by specific RIAs.
Radioimmunochemical assays (RIAs)
The concentrations of insulin, IGF I and C-peptide in tissue
homogenates and culture supernatants were measured by
means of specific RIAs. Insulin was measured according to
Hunter and Greenwood (1982) with a commercial kit (Boris
KidriC Institute). IGF I was measured according to Daughaday etal. (1982) with a commercial kit (Amersham). Cpeptide was measured according to the method of Helding
(1975) using a commercially available kit from Byk Malinckrodt.
Protein concentration
This was measured in tissue homogenates by the method first
described by Lowry et al. (1951).
Statistical analysis
This was performed by the Student's Mest.
Results
Insulin and IGF I activity in embryonic tissue
Fig. 1A shows the level of insulin in embryo homogenates (8- to 12-day-old mouse embryos). In all the tested
samples, measurable amounts of this growth factor
were found, although at this time the /S cells, which are
the only source of insulin in the adults, are not
detectable. Already in the 8-day-old embryos there was
a measurable insulin activity which reached its maximum level in the 9-day-old embryos (277 ^i.u. mg"1
proteins). On the 12th day of gestation, the insulin
activity decreased to 50/a.u.mg~ proteins. The present data are the results of three experiments.
In order to show that the measured activity was the
result of insulin synthesis, we investigated the presence
of C-peptide, the secondary product of this process. As
shown in Fig. 1A (insert), the C-peptide was present in
-o- Insulin
-*• C-peptide
10 11 12 13
of gestation
11
Days of gestation
12
o
I
00
u.
O
4
10
11
Days of gestation
Fig. 1. Insulin (A) and IGF I (B) concentrations in tissue
homogenates of 8- to 12-day-old mouse embryos measured
by specific RIAs. Comparison between insulin and Cpeptide levels in the same samples (A-insert).
all the measured samples. The relation between the
amount of this peptide and insulin was constant, giving
indirect evidence for insulin-derived activity.
In the same samples (8- to 12-day-old mouse embryos), the concentration of IGF I was measured by
specific RIA. As can be seen in Fig. I B , the concentration of IGF I varies between different days of
development in the range of 0.425-1.818 i.u. mg" 1
proteins. The highest values were detected on day 12
(1.818i.u. mg" 1 proteins) and day 9 (1.583 i.u. mg" 1
proteins). The lowest amount of IGF I 0.425 i.u. mg" 1
proteins, was measured on day 10.
Insulin and IGF I activity in primary cultures of
embryonic cells
Indirect evidence for the hypothesis that embryonic
cells from the postimplantation stages of mouse development secrete insulin and IGF I (and/or related
molecules) was provided by an in vitro experiment.
Primary cultures of embryonic cells derived from 8-, 9and 10-day-old mouse embryos were established as
described in Materials and methods. After growing the
cells for 3 days in serum-free conditions, their supernatants were collected and tested for the presence of the
Insulin and IGF I in mouse embryogenesis
493
8 7
§ 0
"
control
8
9
Days in culture
10
B
8 0.15
2
£ o.io
s.
3 0.05u. 0.00 J
O
control
Days of gestation
Fig. 2. Insulin (A) and IGF I (B) concentrations in the
supernatants of cells derived from 8-, 9- and 10-day-old
mouse embryos, and normal mature mouse fibroblasts. In
the supernatants of mouse fibroblasts there were no
measurable insulin activity (A, control).
insulin and IGF I. We found that insulin and IGF I were
present in all samples of conditioned medium.
Insulin
Insulin concentration in the supernatants of cells derived from 8- and 9-day-old mouse embryos was
1.31/ii.u. 10 cells and 0.8jui.u.l0 cells, respectively. Even more insulin activity (5.45^li.u. 10~5cells)
was measured in the culture supernatant of cells derived
from 10-day-old embryos (Fig. 2A). As a control, the
insulin concentration in the supernatant of normal
fibroblasts, cultured under the same conditions was
assessed. On the third day of culture, in the serum-free
medium, there was no measurable insulin activity.
IGF I
IGF I concentration in the medium of mouse embryonic
cells was elevated in comparison to cultures of normal
mouse fibroblasts. The concentration of IGF I in the
culture media of the cells derived from 9- and 10-dayold embryos was 0.109 i.u. per 5xlO5 cells and 0.125 i.u.
per 5xl0 5 cells, respectively. The culture supernatants
of cells derived from 8-day-old mouse embryos contained 0.048i.u. per 5x10^cells IGF I, which is very
close to the concentration in the control medium in
serum-free fibroblasts cultures (0.05i.u. per 5xlO5
cells) (Fig. 2B).
The absence of insulin secretion in control fibroblasts
(Fig. 2A) and the demonstration of IGF I production
by these cells (Fig. 2B) gives additional proof for the
specificity of the radioimmunoassay without any
measurable cross-reactivity of insulin and IGF I.
Fig. 3. The number of cells in cultures derived from 9-dayeither
old mouse embryos 13 days after.the addition of
insulin (10/.u.xi. ml" ) (A) or IGF I (lOngml"1) (B).
The influence of insulin and IGF I on primary cultures
of embryonic cells
We further examined whether insulin and IGF I can
stimulate the proliferation of mouse embryonic cells in
vitro.
Since our previous results have shown that the tissue
homogenates of 9-day-old embryos contain the highest
concentration of IGF I and insulin, these embryos were
selected for the further analysis. The cells derived from
9-day-old embryos were cultured in serum-free conditions. Either insulin or IGF I was added to these
cultures at physiological concentrations, 10 i.u. ml" 1
and lOngml"1, respectively. 3 days later the cells were
counted. The number of cells was significantly higher in
the cultures with either insulin or IGF I as compared to
the control cultures where cells were grown without the
investigated growth factors (Fig. 3). The number of
cells in the group where insulin was added was
47 000 cells ml" 1 (P<0.02) and in the one where IGF I
was added 45000cellsmr 1 (P<0.01). In the control
group there were 22 000 cells ml"1.
Discussion
It is still not clear whether embryonic cells in the early
stages of development secrete insulin and if this peptide
stimulates their proliferation. However, there is some
evidence that insulin has a physiological role during
embryogenesis. Namely, it has been shown that insulin
stimulates growth of embryonic cells in vitro. Furthermore, De Pablo et al. (1982) demonstrated the presence
of immunoreactive insulin in prepancreatic, 2-day-old
chicken embryos which was required for their development (De Pablo et al. 1985). Miller (1988) has reported
that stage IV oocytes from Xenopus laevis increase
RNA, protein and glycogen synthesis in response to
extracellular insulin. In addition, a similar role of
insulin was also shown for some embryonal carcinoma
(EC) cell lines, which can, in spite of their tumorogenous nature, serve as an experimental model for the
investigation of postimplantation stages of development (Martin, 1980). Physiological concentrations of
insulin stimulates proliferation of embryonal carcinoma
cell line F9 by binding to its own receptors (Nagarajan
and Anderson, 1982). Undifferentiated PC13 EC cells
do not express insulin receptors, but their differentiation in the presence of the retinoic acid results in the
494
R. Spaventi, M. Antica and K. Pavelid
appearance of specific surface binding sites over which
insulin increases DNA synthesis and proliferation
(Heath et al. 1981). Another cell line, teratoma-derived
line 1246-3A, produce a polypeptide growth factor
tentatively called IRF (insulin-related factor), which is
structurally and biochemically similar to pancreatic
insulin but different from IGF I and IGF II (Yamada
and Serrero, 1988). The same authors showed that IRF
acts as an autocrine growth factor for these cells.
Since teratoma and teratocarcinoma cell lines produce several hormones and growth factors that have
also been found in the embryo (D'Ercole et al. 1980;
Adamson, 1982; Rizzino, 1985), it is reasonable to
assume that insulin secreted by them may also be a part
of the physiological growth regulatory system during
embryonic development. One explanation of these
findings is that in the pluripotent cells the insulin gene is
active, while later, after differentiation and specialization, it remains active only in some differentiated
tissues like the /S cells of the pancreas, the yolk sac and
possibly liver of the rat embryos (Muglia and Locker,
1984; Kimberley etal. 1989).
Recent results indicate that insulin is necessary for
embryonic development from the early-somite stage
(Travers et al. 1989). This hormone is also important for
the normal growth of mouse conceptuses from the 2-cell
embryo to the blastocyst (Rappolee et al. 1989; Harvey
and Kaye,' 1989). The discovery of active insulin receptors at these same stages of development support these
results (Untermann etal. 1986). Since it is known that
insulin does not cross the placenta (Adam et al. 1969;
Kalhan and Adam, 1975) and that insulin secretion
from the yolk sac starts about the same time as pancreas
is developed (14th day in the rat) (Muglia and Locker,
1984; Kimberley et al. 1989), the question is what is the
source of this peptide before that period? None of the
results give a conclusive answer to this question.
In the present study, we show that insulin is secreted
by non-extraembryonic mouse tissues. We found that
there are measurable amounts of insulin in the embryonic tissues of 8 j to 12-day-old mouse embryos. Since
the /3 cells, which are the only source of insulin in the
adult mouse, could be detected on the 11th day of
gestation (Rail etal. 1973), the insulin detected before
day 11 could be defined as extrapancreatic. The presence of this 'extrapancreatic' insulin was confirmed by
measuring the levels of C-peptide, which is an indirect
measure of normal insulin synthesis. Insulin detected
on the 11th and 12th day could also be of pancreatic
origin.
Further support for the hypothesis that embryonic
cells secrete insulin at these early stages of embryogenesis was provided by in vitro experiments. The cells
from primary cultures derived from 8-, 9- and 10-dayold mouse embryos secrete insulin and/or some related
molecules. There is the possibility that some of these
cells differentiate during the cultivation period and
therefore secrete insulin. However, although it is
known that whole embryos in culture could differentiate to a certain degree (New, 1978; Sadler, 1979), this
is less likely for the cell suspensions because of the
different milieu and growth conditions. This is supported by the cell morphology at the end of the
cultivating period (data not shown).
Since the 9-day-old embryos contain the highest
concentration of insulin, we tested the effect of exogenous insulin on the number of primary cultured embryonic cells. We show that physiological concentrations of
insulin increase the number of cells significantly more
than in the control cultures (embryo cells grown in
unsupplemented serum-free medium).
These results lead us to the conclusion that insulin is
secreted during the period of early embryogenesis when
pancreas is not yet formed by the embryonic cells
themselves, and that it might play an important role in
their growth regulation.
IGF I is a polypeptide thought to be involved in the
stimulation of somatic growth. Although its role in
embryogenesis has not been elucidated, it has been
shown that mouse fetal tissues at the stage of organogenesis (9-12 days of gestation) possess cell membrane
receptors of relatively high affinity for IGF I (Smith
etal. 1987). Also, the embryonal carcinoma cells
(PC13) express IGF receptors and secrete binding
protein into the medium (Heath and Shi, 1986; Smith
etal. 1987). D'Ercole and co-workers (1980) investigated the production of IGF I in mouse embryo organ
explants. They found that the intestine, heart, brain,
kidney, liver and lung, but not the placenta, of 17-dayold mouse embryo secrete immunoreactive IGF I in
vitro. In the same study they detected this growth factor
in the media of fetal liver explants from day 11
embryos.
In this paper, we give evidence that IGF I is produced
in the mouse embryo as early as the 8th day of
gestation. The concentration of IGF I in media in which
the embryonic cells were cultured was significantly
higher than in the culture containing control, mature
fibroblasts.
It seems that the synthesis and release of peptide
growth factors, among them insulin and IGF I, might be
the property of most, if not all, embryonic cells and that
they act locally like autocrine and paracrine, but not as
endocrine growth signals.
The authors would like to thank Drs Lidija Suman, J.
Pavelid, S. MaruSid-GaleSid and S. Levanat for critical evaluations of the manuscript.
This research has been undertaken with a financial contribution by the Commission of the European Communities
Directorate-General for Science, Research and Development
International Scientific Cooperation.
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(Accepted JO November J989)