| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Institut National de la Santé et de la Recherche Médicale, Unité de Recherches sur la Régulation de la Croissance, Hôpital Saint Antoine, Paris 75571, France
Address all correspondence and requests for reprints to: M. Binoux, INSERM Unité 142, Hôpital Saint Antoine, 184, rue du Faubourg Saint Antoine, 75571 Paris Cedex 12, France.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1-antitrypsin promoter so
as to obtain liver-specific expression. Five transgenic founder mice were raised, only two of which (lines 124 and 149) produced transgenic offspring. Northern blotting revealed transgene expression exclusively in the liver during fetal life and unchanged through to adulthood, whereas expression of the endogenous gene was undetectable beyond 10–15 days postnatally. hIGFBP-1 was detected by western immunoblotting in the plasma of transgenic mice and IRMAs yielded mean concentrations of 2.41 ± 0.33 ng/ml and 13.69 ± 1.42 ng/ml in homozygous animals of lines 124 and 149, respectively. In the latter, IGFBP-1 levels were distinctly higher than in heterozygotes (2.99 ± 0.39 ng/ml), P < 0.0001. These levels remained stable in each given animal and did not change with age. Plasma concentrations of IGF-I measured in line 149 exhibited the well-known profile of an increase from birth up to puberty. Values for heterozygotes were similar to those for wild-type mice, with adult levels (544 ± 98 ng/ml) slightly below those of controls (630 ± 56 ng/ml), P < 0.05. In homozygotes they were distinctly lower, with adult levels of 370 ± 75 ng/ml, P = 0.001. In heterozygous and homozygous adults, there was a negative correlation between IGF-I and IGFBP-1 concentrations (r = 0.8, P < 0.0001), suggesting a link between transgene expression and IGF-I levels.
Study of body weight gain in line 149 revealed growth retardation within the first weeks after birth, which was marked in homozygous males and females (P < 0.001) but also present in heterozygous males (P = 0.002), indicating some relationship with transgene expression. In addition, body weight in adult mice was negatively correlated to plasma concentrations of IGFBP-1 (r = 0.7, P < 0.0001).
Reproductive function also appeared to be severely affected, especially in homozygous females: mating that failed to result in pregnancy in half of the homozygous females crossed with nontransgenic males, suggestive of impaired fertilization or implantation; interrupted or prolonged pregnancies with fetal and neonatal death. Litter size was reduced in transgenic females (by about half in homozygotes) and in nontransgenic females mated with homozygous males, resulting from pre- or neonatal mortality. Moreover, deaths occurred within the first 5 days of life, with an incidence of approximately 50% in the litters of homozygous females, 12–18% among heterozygotes mated with nontransgenic or heterozygous males, respectively, and 30% among those mated with homozygous males. These results, suggesting that fetal transgene expression largely accounted for ante- and perinatal mortality, were confirmed by the predominance of homozygotes among those that could be analyzed genetically. Similarly impaired reproductive function was seen in line 124, but to a lesser degree.
Although the mechanisms responsible for these disorders remain to be determined, our results indicate that permanent and uncontrolled hepatic expression of IGFBP-1, even at low levels, affects fertility in females and both ante- and postnatal development.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In all biological fluids, IGFs are noncovalently bound to high affinity (109–1011 M-1) binding proteins, (IGFBP-1 to -6). Like the IGFs, they are synthesized ubiquitously, but each is subject to different tissue-specific and developmental regulation. They control the bioavailability of the IGFs and modulate interactions between the IGFs and their target cells (2, 3).
IGFBP-1 is produced primarily in the liver and decidualized stromal cells of the endometrium (3, 4). Hepatic production is maximal during fetal life, decreases rapidly after birth, and thereafter is dependent on insulin secretion that inhibits its synthesis at the transcriptional level (5). The abundant endometrial expression of IGFBP-1 early in gestation (6) suggests that it is involved in local control of cell proliferation. Depending on its state of phosphorylation and the cell system involved, IGFBP-1 may either potentiate or inhibit IGF action (2, 4, 6). It has also been shown to act independently of its binding to IGFs (7). However, its physiological role in growth and metabolism remains poorly understood.
The aim of this study was to investigate the endocrine action of
hepatically produced IGFBP-1 using transgenesis in which human IGFBP-1
(hIGFBP-1) cDNA was placed under the control of the liver-specific
1-antitrypsin promoter. While this work was under way, two reports
of transgenesis were published, in which the IGFBP-1 gene was placed
under the control of ubiquitous promoters, i.e.
metallothionein (8) and phosphoglycerate kinase (9). We describe here
our two lines of hIGFBP-1 transgenic mice and some of the effects of
liver-specific, long-term overexpression of IGFBP-1 on growth and
development.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The IGFBP-1 cDNA was then cloned to the HindIII site of a
pBluescript vector containing the 700 bp of the human 1-antitrypsin
promoter (gift from R. Cortese, IRBM, Rome, Italy) so as to obtain
predominantly liver-specific expression (10) (Fig. 1A
).
|
1-antitrypsin promoter (11)
(gift from M. Yaniv, Institut Pasteur, Paris, France). Western ligand
blot analysis of the IGFBPs in media conditioned by these cells
confirmed that our construct was functional (Fig. 1B
).
Microinjection of the insert
The insert was excised from its plasmid by double digestion with
XhoI/NotI (Fig. 1A), then purified by agarose gel
electrophoresis and chromatography on Elutip-d columns (Schleicher &
Schuell, Dassel, Germany). The purified insert was taken up in 10
mM Tris, 0.1 mM EDTA microinjection buffer.
Microinjection was carried out as previously described (12) into fertilized ovocytes of C57BL6/DBA2 F2 hybrid mice, pretreated with 5 U PMS, then 5 U human CG (hCG) to obtain superovulation. Ova having survived microinjection were then transferred into the oviducts of pseudopregnant females.
Animals and biological samples
Mice were bred in our animal house. They were fed with lab chow
and submitted to a regulated light cycle (12-h light, 12-h
darkness).
Wild-type mice were B6/CBA animals.
Blood samples were collected in 0.13 M EDTA. Samples were taken by intracardiac puncture in fetal and newborn animals and intraorbital puncture in adults. After centrifugation, plasma samples were divided into aliquots and stored at -20 C.
Animals were killed by cervical dislocation and the liver and other organs immediately frozen in liquid nitrogen and stored at -70 C for RNA extraction.
All protocols used were approved by institutional review committees.
Extraction of genomic DNA, Southern blot, and PCR analyses
Tail fragments were cut at 3 weeks of age and digested overnight
with 400 µg proteinase K in 50 mM Tris-HCl, pH 7.5, 50
mM EDTA, 100 mM NaCl, 1% SDS, 5 mM
dithiothreitol, and 0.5 mM spermidine at 56 C. High mol wt
DNA was isolated by extraction with phenol-chloroform and precipitated
in 100% ethanol (13).
After identification of founder transgenic mice using Southern blot
analysis, subsequent generations of transgenic progeny were identified
by PCR analysis of 1 µg of genomic DNA using HI-TAQ polymerase
(Bioprobe, Montreuil, France). Synthetic oligonucleotides (Institut
Pasteur, Paris, France) used for PCR were designed to amplify a
384-bp fragment of the transgene. The oligomers were:
5'-GCCAGGTACAATGACTCCTTTCGG-3' that hybridizes with the
1-antitrypsin promoter sequence and 5'-GGACCTCTGACATCTCCAGGC-3' that
hybridizes with a sequence in exon 1 of the cDNA coding for hIGFBP-1.
Heterozygous and homozygous transgenic animals were identified on the
basis of Southern blot analysis of DNA samples.
For Southern blot analysis, 10 µg mouse genomic DNA were digested overnight with restriction endonucleases at 37 C (New England Biolabs, Beverly, MA), fractionated on 1% agarose gel and transferred to Gene Screen Plus membranes (DuPont-New England Nuclear Research Products, Boston, MA), according to the Southern method (14). After 2 h of prehybridization at 42 C in 50% formamide, 5 x SSC, 50 mM phosphate buffer, pH 6.5, 1% SDS, 5 x Denhardt’s and 100 µg/ml sonicated salmon sperm DNA, the blots were hybridized for 20 h at 42 C with the same hIGFBP-1 cDNA fragment as that used to construct the fusion gene labeled by random priming (specific activity: 1–2 x 106 cpm/ng) using the Megaprime DNA labeling system (Amersham, Little Chalfont, Buckinghamshire, UK). The hybridization buffer was the same as that used for prehybridization, plus 5% dextran sulphate. The blots were washed twice for 15 min in 2 x SSC, 0.1% SDS at room temperature and once for 30 min in 0.1% SSC, 0.1% SDS at 65 C, then autoradiographed at -80 C with intensifying screens.
Isolation of RNA and Northern blotting
Total RNAs were extracted from frozen livers using the standard
CsCl/guanidine isothiocyanate method (15). Forty micrograms of total
RNA were loaded onto 1.2% agarose/8% formaldehyde gels, submitted to
electrophoresis in MOPS 1x, transferred to Hybond-N nylon membranes
(Amersham) and covalently bound to the nylon by baking of the membranes
at 80 C for 2 h. After prehybridization at 42 C in 50% formamide,
5 x SSPE, 1% SDS, 5 x Denhardt’s and 100 µg/ml
sonicated salmon sperm DNA, the blots were hybridized for 20 h at
42 C in the same buffer, plus 5% dextran sulphate, to 2.5 x
106 cpm/ml hIGFBP-1 cDNA probe (SA: 1–2 x
106 cpm/ng). The blots were washed twice for 15 min in
2 x SSPE, 0.1% SDS at room temperature and once for 30 min in
0.1% SSPE, 0.1% SDS at 65 C, then autoradiographed at -80 C with
intensifying screens.
Western ligand blotting and immunoblotting
Plasma samples were analysed by western ligand blotting
according to Hossenlopp et al. (16). Briefly, 3 µl of each
sample were submitted to 12.5% PAGE under nonreducing conditions.
Serum proteins were electrotransferred onto nitrocellulose membranes
that were then incubated with a mixture of 125I-IGF-I and
-II. IGFBPs were revealed by autoradiography.
The nitrocellulose membranes used for ligand blotting were then prepared for immunoblotting. In this case, IGFBP-1 bound to the nitrocellulose membrane was revealed using a 1/1 000 dilution of rabbit polyclonal antirecombinant human IGFBP-1 (rhIGFBP-1) antibody generously provided by Kabi (Stockholm, Sweden). The antibody proved to be specific for IGFBP-1, although it cross-reacted with murine IGFBP-1. After incubation with goat polyclonal antirabbit IgG antibody coupled to horseradish peroxidase, IGFBP-1 was detected using the Amersham ECL Western blotting detection system.
Other assays
hIGFBP-1 assay. Human IGFBP-1 was measured in plasma samples
using an IRMA specific for human and primate IGFBP-1, with no
cross-reaction with murine IGFBP-1 (DSL 7200 Active IGFBP-1 IRMA Kit,
Diagnostic Systems Laboratory, Webster, TX). Each sample was studied at
two concentrations (30 µl and 15 µl plasma per assay tube). The
sensitivity threshold was 0.25 ng/ml plasma. No immunoreactive IGFBP-1
was detectable in wild-type mice.
IGF-I assay. Plasma samples (25 µl) were incubated in acid medium (0.01 M HCl) for 30 min at room temperature to dissociate IGFs from IGFBPs, then ultrafiltered on Centricon 30 (Amicon, Epernon, France) to separate IGFs from IGFBPs (Y. Le Bouc et al., Hôpital Trousseau, Paris, France, manuscript in preparation). The ultrafiltrate containing IGFs was lyophilized, then taken up in 0.1 M phosphate buffer, 1 mg/ml BSA, pH 7.4, and incubated for 2–3 days in a final volume of 400 µl with a specific polyclonal antihuman IGF-I antibody (1:120 000 dilution) that cross-reacts with murine IGF-I (gift from F. Frankenne, Centre Hospitalo-Universitaire de Liège, Belgium) and 125I-hIGF-I (3 000 cpm/tube). Iodination was performed using the chloramine-T method. Unknown samples were tested at 3 concentrations plus one blank (without antibody), each in duplicate so as to confirm parallelism with the standard curve. After incubation, free and bound IGFs were separated using albumin-coated charcoal, as previously described (17). The threshold sensitivity of the assay was 1–2 ng/ml plasma. Intraassay variation was close to 5% and interassay variation, 10%.
Proteinuria. Proteinuria was detected and semiquantitatively estimated using Multistix 8 SG strips (Bayer Diagnostics, Puteaux, France).
Glycaemia. Reactive strips (Glucotides, Bayer Diagnostics) and a glycaemia reader (Glucometer 4, Bayer Diagnostics) were used to measure glucose levels.
Statistical analysis
Classical methods including ANOVA were used to study differences
between groups. Tests were considered significant at P
< 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
).
|
), strong signals corresponding to the
expected size (2.3 kb) were obtained for Mice 146 and 149. In the other
mice, the insert sizes were either larger (nos. 124 and 162) or smaller
(no. 13) than expected (Fig. 2A).
With TaqI, all animals except no. 162 yielded two strong
bands corresponding to DNAs of sizes that together were equal to the
size of the insert (Fig. 2B).
The integrity of the transgene was checked in all animals using a restriction endonuclease cleaving at a site different from that of the insert (data not shown). A single signal was obtained in each case, which also demonstrated that the transgene had been integrated at a single site in the host genome.
The founder mice were then crossed with nontransgenic animals, but only Nos 124 and 149 produced transgenic progeny. Further studies therefore concerned these two lines.
The number of copies of the transgene integrated was determined for each line. In the case of line 124, a single copy and, in the case of line 149, four copies were integrated at the single site in the host genome.
Hepatic expression of the transgene
Because the promoter used was liver-specific, we first
concentrated on Northern blot analysis of hepatic transgene expression
as related to age. Total RNA samples extracted from livers of animals
killed at days 15 and 19 in utero and at intervals between
birth and adulthood were analysed.
For line 149, signals were obtained at 1.65 kb, confirming that
transgene expression was hepatic (Fig. 3). Although
endogenous mouse IGFBP-1 mRNA is of a similar size (1.6 kb) to that of
the transgene messenger (1.65 kb), Fig. 3 shows that homozygous
transgenic animals yielded a stronger signal than nontransgenic
controls. There was therefore strong expression of hIGFBP-1 in the
former. This overexpression was marked during the perinatal period and
still detectable 90 days postnatally, whereas in controls expression
was no longer detectable beyond 10–15 days.
|
), although transgene expression was weaker but
still detectable through to adulthood.
|
Tissue specificity of transgene expression was checked by analysing IGFBP-1 mRNA in the kidneys, testes, ovaries, and uteri at postnatal days 18 and 30 and at adulthood. No difference was found between transgenic mice of either line and nontransgenic controls (data not shown).
Detection of hIGFBP-1 in the bloodstream
Blood samples were taken from both lines during fetal (19 days
in utero) and postnatal (from birth to adulthood) life and
analyzed by Western ligand- and immunoblotting.
Ligand blotting of plasma samples taken from line 149 during the
perinatal period revealed a major IGFBP migrating around 30 kDa in both
transgenic and control animals (Fig. 5A). The signal
gradually faded with age, at the same time as the characteristic 42- to
39-kDa doublet corresponding to IGFBP-3 appeared. Immunoblotting using
anti-IGFBP-1 antibody confirmed that the 30-kDa band corresponded
to IGFBP-1. The signal was strong during the perinatal period (19 days
in utero through to postnatal day 3) in both transgenic and
control animals (because the antihuman IGFBP-1 antibody used
cross-reacts with murine IGFBP-1) (Fig. 5B), although stronger in
the former. After day 6, the signal was weaker but persisted in
transgenic animals, whereas in controls it was barely or no longer
detectable. Similar results were obtained for line 124 (not shown).
|
shows plasma concentrations of hIGFBP-1 measured
by IRMA. For homozygous animals of line 149, these were between 4.9 and
25 ng/ml; for line 124, between 0.3 and 5.5 ng/ml. The difference
between the two lines was highly significant (P =
0.0001). Heterozygotes had lower IGFBP-1 levels than homozygotes,
reflecting weaker transgene expression in the liver: 0.3–4 ng/ml for
line 124 (P = 0.05); 0.96–7.7 ng/ml for line 149
(P < 0.0001).
|
Plasma IGF-I levels
Like plasma IGFBP-1, circulating IGF-I is produced in the liver,
and it was therefore important to check plasma concentrations of
IGF-I.
In line 149, transgenic animals and controls, IGF-I levels typically
increased with age, with the well-known peak during puberty and
stabilization of values at a lower level at adulthood between 200 and
500 ng/ml (Fig. 6). Heterozygote levels (544 ± 95
ng/ml) were slightly lower than those for controls (630 ± 56
ng/ml), P < 0.05. In homozygotes, levels were more
significantly lower (370 ± 75 ng/ml), P = 0.0001.
The difference was more obvious during the growth period, but our data
were too limited for statistical analysis. The lowest plasma IGF-I
levels (<50 ng/ml before 6 weeks and <250 ng/ml at 60 days and older)
were obtained for animals with the most marked growth retardation.
|
).
|
).
|
). Covariance analysis of the all data for day 10 through
to day 120 revealed significant differences, both in females
(P < 0.0001) and in males (P <
0.0001). For heterozygotes, covariance analysis revealed growth
retardation among males (P = 0.002), but not females,
although the curves for mean weight gain appeared superimposable with
those for controls.
Interestingly, body weight in transgenic adult animals was
negatively correlated (r = 0.7, P < 0.0001) to
plasma hIGFBP-1 levels (Fig. 9).
|
Reproduction, litter size, ante- and perinatal mortality
Mating did not always result in pregnancy (Table 2). Among transgenic females, this was most evident
in the case of homozygotes, where, on average, the success rate was 1
in 2 when paired with nontransgenic or heterozygous males. Fertility in
heterozygotes appeared to be normal, 21 out of 22 pregnancies occurring
after mating with nontransgenic males, and 67 out of 76 with
heterozygous males. Male heterozygote fertility also appeared normal in
view of the success rate with heterozygous females and, in addition, 19
out of 20 pregnancies were achieved after mating with nontransgenic
females. However, this was not necessarily true for homozygous males,
where the success rate was 14 out of 18 with nontransgenic females, 9
out of 13 with heterozygotes and 1 in 5 with homozygous females.
|
). Nineteen
female heterozygotes were killed at between 8 and 18 days of gestation.
In 16 cases where weight gain had been normal, a normal number of live
fetuses was found. In the three remaining animals, where weight gain
had been insufficient, smaller numbers of fetuses were found and
several were dead. The global incidence of interrupted pregnancy was
17%, with no preponderance in any particular group (Table 2). Mating
of homozygous males with heterozygous females resulted in the same
number of interrupted pregnancies as did mating with nontransgenic
females.
|
). Pregnancies prolonged to 20 or 21 days were observed in 8%
of wild-type animals, but in 20% of wild-type/heterozygote pairings,
in 40% of heterozygote/heterozygote pairings and in 75% of
heterozygote/homozygote pairings. Reduced litter size. The mice normally gave birth at night, which meant that litters were counted several hours later (15–18 at most). Average litter size was 8 in controls. In line 149 animals, it was reduced to around 6 in heterozygotes and in wild-type females mated with homozygous males, and to around 4 when heterozygotes were mated with homozygotes. It was even further reduced in cases of prolonged pregnancy. In the one pregnancy to reach term after homozygote/homozygote mating, no pups were found in the cage. These observations do not provide information on litter size in utero; they correspond to the number of pups that survive the first hours of life, because the females normally eat stillborns.
In line 124 animals, litter size was normal for heterozygous females (around 8). In contrast with line 149, 15 homozygote/homozygote pairings led to 14 full-term pregnancies. For 12 of these, there was a mean of 4.4 pups/litter; in the remaining 2, no pups were found.
Mortality within the first days of life. After the
initial newborn litter counts, deaths were observed (either dead pups
or pups disappeared) over the first 5 days. The data in Table 3 clearly show the increasing percentages of deaths
depending on whether wild-type females were mated with transgenic
males, or heterozygous females with heterozygous or homozygous males,
or homozygous females mated with wild-type or heterozygous males. Among
heterozygous/heterozygous pups, the largest number bred, genomic DNA
samples could be analyzed for 16 dead within the first 5 days. Four
were nontransgenic, 2, heterozygous and 10 homozygous. The homozygotes
therefore represented two-thirds of the dead pups, whereas
theoretically, one in four offspring of heterozygous/heterozygous pairs
would be homozygous.
|
Glucose tolerance tests done on transgenic line 149 animals suggest that in homozygotes, the return to basal glucose levels is comparatively slow. The effect was apparently present, but less marked, in heterozygotes. These findings await confirmation.
Proteinuria (1–5 g/liter) was detected at 3–5 months of age in 2 out of 21 heterozygotes and in 7 out of 19 homozygotes. Histological examination revealed normal kidneys in three nonproteinuric heterozygotes, but lesions of the glomerulus in three proteinuric homozygotes, which are currently under more detailed study.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1-antitrypsin promoter. Initial studies have been undertaken of the
repercussions of the resulting constant levels of hIGFBP-1 protein in
the bloodstream. In normal circumstances, circulating IGFBP-1 is
essentially of hepatic origin (5) and these lines of transgenic mice
constitute a good model to investigate its endocrine action in the
absence of physiological regulatory factors. Northern blotting confirmed that transgene expression was liver-specific and permanent, whereas in control animals IGFBP-1 expression was maximal during the final days of fetal life and perinatally, thereafter becoming undetectable by this technique beyond the age of 2 weeks. This age-related profile is common to numerous species and, in particular, identical in the rat (18). Transgene expression was demonstrated by both Northern blotting and measurement of plasma protein levels that remained stable with age. Predictably, it was stronger in homozygotes than in heterozygotes. It was also found to be significantly more marked in line 149 than in line 124, which was the reason for our concentrating on line 149. An important observation, in view of the severe disorders noted among transgenic animals, was that plasma levels of IGFBP-1 were on average below 5 ng/ml in heterozygotes, but 15 ng/ml in homozygotes.
Because IGF and IGFBP synthesis in the liver are closely linked, possible effects of hepatic expression of the transgene on that of IGFs and IGFBPs needed to be checked. In the case of IGFBP-1, neither Northern nor Western blotting would be informative because the electrophoretic migration of the murine and human forms is very similar and no RIA specific for murine IGFBP-1 has yet been developed. As regards the other serum IGFBPs, Western ligand blot analysis revealed no differences between transgenic and control mice.
Serum levels of IGF-I were found to increase with age up to puberty, as in most mammals (19, 20), with similar concentrations in heterozygous and control animals and generally lower concentrations in homozygotes where, at adulthood, the differences from controls and heterozygotes were significant. In the light of present data, it is only possible to speculate on the reasons for these lower concentrations. Sequestration by IGFBP-1 does not seem probable. Unlike the 140-kDa complexes that transport most of the IGF-I in the bloodstream (21, 22), IGFBP-1 is capable of crossing the capillary endothelium. Even if a fraction of the IGF-I secreted by the liver binds to IGFBP-1 and is thereby more rapidly cleared, it is unlikely that this would significantly alter total serum concentrations, particularly in view of the low levels of hIGFBP-1 in transgenic mice compared with those of IGF-I or IGFBP-3 (as estimated by Western ligand blotting). Another hypothesis would be some effect on hepatic synthesis of IGF-I that is GH-dependent (1). We have no data for GH in these transgenic mice but it would seem unlikely that GH secretion was depressed. It would more probably be increased as a result of hypothalamus and pituitary activation, if the excess IGFBP-1 blocked IGF-I mediation of GH negative feedback (23). IGFBP-1 could also directly inhibit hepatic IGF-I synthesis, both peptides being produced by hepatocytes (1, 3). The negative correlation observed between IGF-I and IGFBP-1 levels in adult hetero- and homozygous animals is pertinent here, clearly demonstrating the link between IGF-I levels and transgene expression.
Measurement of body weight gain with age revealed significant growth retardation in homozygous males and females and to a lesser extent in heterozygous males. In many cases, this became evident after the age of 2 weeks, although in some animals it appeared earlier. Apart from the possible effects of the reduced IGF-I levels mentioned above, the stunted somatic growth may also be caused by IGFBP-1-induced inhibition of the mitogenic action of IGF-I, in that there was a dosage-related effect, the homozygotes exhibiting the more severe growth retardation and having the higher levels of IGFBP-1 expression. In addition, there was a negative correlation between adult body weight and hIGFBP-1 levels. It has been shown that IGFBP-1 administered to GH- or IGF-I-treated hypophysectomized rats reduces the effects of these growth stimulators on somatic growth (24). Similar results to ours have been reported by Rajkumar et al. (9). These authors also noted reduced birth weight, which may reflect the fact that their construct was placed under the control of the ubiquitous phosphoglycerate kinase promoter. IGFBP-1 produced ectopically in a variety of tissues may have had stronger effects than in our animals where IGFBP-1 was provided solely by the bloodstream. By contrast, Dai et al. (8), who used the ubiquitous metallothionein promoter, observed no growth retardation despite the significantly higher plasma IGFBP-1 concentrations in their mice than in ours. Nevertheless, our findings are consistent with those for transgenic mice carrying null mutation of the IGF-I gene (IGF-I -/-) where growth retardation occurs essentially after birth (25, 26).
Our data, which clearly demonstrate the links between hepatic expression of the transgene, circulating IGFBP-1 levels and growth retardation, therefore provide strong evidence that the effects observed are unrelated to the site of insertion of the transgene into the host genome. An important aspect of our findings concerns disorders of reproductive function and fetal and neonatal mortality.
Although the fertility of transgenic males appeared normal (at least in heterozygotes), the percentage of matings resulting in pregnancy, which was close to 100% among heterozygous females mated with nontransgenic males, was reduced to 50% among homozygous females, suggesting impaired fertilization or implantation and/or precocious abortion. It has been demonstrated that neither the IGFs nor the type 1 IGF receptor are of vital importance at the beginning of embryonic development (26, 27) and abortion resulting from abnormal development at this early stage therefore seems unlikely. However, several studies have provided data in support of the IGFs and IGFBPs, either provided via the circulation or produced locally in the granulosa, being involved in follicular maturation and selection for either ovulation or atresia (6, 28, 29). Furthermore, IGFs are mediators of oestradiol action on proliferation of the uterine endometrium (30). Maternal IGFBPs play a role in regulating the effects of IGFs involved in blastocyst implantation. IGFBP-1, which is strongly expressed in the stromal cells of the decidualized endometrium, would serve to limit IGF-stimulated growth of trophoblastic cells and prevent trophoblast invasion during blastocyst implantation (27, 31). It therefore seems that in our transgenic mice the excess of IGFBP-1 in the bloodstream could adversely affect follicular maturation or implantation. In the transgenic model of Rajkumar et al., where IGFBP-1 was overexpressed in numerous tissues, including the uterus, impaired fertility was also observed in homozygous females (9) and the uterine response to estradiol in these mice was markedly depressed (32). The infertility reported in IGF-I -/- mice (26) would also corroborate our findings.
Impaired fetal development was suggested by the incidence of interrupted pregnancies (on average, 17%) that was as high among transgenic as control females when crossed with homozygous males. Fetal transgene expression was therefore directly involved. Intrauterine death, as suggested by stabilization or loss of weight, was demonstrated in several cases, whereas in females where weight gain was normal, the size and number of fetuses were normal up to day 18 in utero.
Prolonged pregnancies also occurred, and more frequently in the types of cross where the probability of transgenic progeny was greater. In all prolonged pregnancies, there was a higher incidence of neonatal mortality, reaching around 50% in the case of hetero-/homozygous pairs.
Apart from intrauterine and neonatal deaths, there was increased mortality within the first 5 days of life. The incidence was of approximately 50% in the litters of homozygous females, 12% and 18% among heterozygotes mated with nontransgenic or heterozygous males, respectively, and 30% among those mated with homozygous males. The deaths at birth or within 5 days postnatally concerned predominantly transgenic (mostly homozygous) but also nontransgenic pups, which suggests impaired parturition or lactation that may result in the deaths of the pups. The prolongation of pregnancy observed would also support the notion of impaired parturition.
The repercussions on somatic growth and reproductive function seen in line 149 were also noted in line 124, albeit to a lesser degree, in agreement with the weaker hepatic expression of the transgene and lower plasma concentrations of IGFBP-1. Southern blot analysis of genomic DNA of each founder animal revealed different transgene insertion sites and different numbers of inserted copy for each line. From this and the relationships observed between the number of copies integrated, the extent of transgene expression and the severity of the phenotype anomalies, we conclude that the effects were attributable to expression of the transgene and not to mutagenic insertion.
Deletion of the type 1 IGF receptor gene has been shown to be consistently lethal to the fetus at birth (26). Deletion of the IGF-I gene results in 95% mortality within the first two weeks of life, accompanied by severe developmental abnormalities in many tissues and organs (25, 26, 33). In some of our transgenic fetuses, the excess IGFBP-1 may then inhibit the action of IGFs produced locally in vital organs, resulting in development that is incompatible with survival. It is pertinent to note that strong IGFBP-1 expression has been observed in association with intrauterine growth retardation in several species (34, 35, 36, 37).
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received December 16, 1996.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
5ß1 integrin by means of its
Arg-Gly-Asp sequence. Proc Natl Acad Sci USA 90:10553–105571 antitrypsin gene. Cell 41:531–540[CrossRef][Medline]
1-antitrypsin gene. Mol Cell Biol 8:4362–4369This article has been cited by other articles:
 |
|
 |
|
  Y. Zha, V. T. Le, Y. Higami, I. Shimokawa, T. Taguchi, and M. S. Razzaque Life-Long Suppression of Growth Hormone-Insulin-Like Growth Factor I Activity in Genetically Altered Rats Could Prevent Age-Related Renal Damage Endocrinology, December 1, 2006; 147(12): 5690 - 5698. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Shukla, X. Coumoul, L. Cao, R.-H. Wang, C. Xiao, X. Xu, S. Ando, S. Yakar, D. LeRoith, and C. Deng Absence of the full-length breast cancer-associated gene-1 leads to increased expression of insulin-like growth factor signaling axis members. Cancer Res., July 15, 2006; 66(14): 7151 - 7157. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Marchand, C. Tomkiewicz, L. Magne, R. Barouki, and M. Garlatti Endoplasmic Reticulum Stress Induction of Insulin-like Growth Factor-binding Protein-1 Involves ATF4 J. Biol. Chem., July 14, 2006; 281(28): 19124 - 19133. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. S. Watson, P. Bialek, M. Anzo, J. Khosravi, S.-P. Yee, and V. K. M. Han Elevated Circulating Insulin-Like Growth Factor Binding Protein-1 Is Sufficient to Cause Fetal Growth Restriction Endocrinology, March 1, 2006; 147(3): 1175 - 1186. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kajimura, K. Aida, and C. Duan Understanding Hypoxia-Induced Gene Expression in Early Development: In Vitro and In Vivo Analysis of Hypoxia-Inducible Factor 1-Regulated Zebra Fish Insulin-Like Growth Factor Binding Protein 1 Gene Expression Mol. Cell. Biol., February 1, 2006; 26(3): 1142 - 1155. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Stefan, H. Ji, R. A. Simmons, D. E. Cummings, R. S. Ahima, M. I. Friedman, and R. D. Nicholls Hormonal and Metabolic Defects in a Prader-Willi Syndrome Mouse Model with Neonatal Failure to Thrive Endocrinology, October 1, 2005; 146(10): 4377 - 4385. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Marchand, C. Tomkiewicz, J.-P. Marchandeau, E. Boitier, R. Barouki, and M. Garlatti 2,3,7,8-Tetrachlorodibenzo-p-dioxin Induces Insulin-Like Growth Factor Binding Protein-1 Gene Expression and Counteracts the Negative Effect of Insulin Mol. Pharmacol., February 1, 2005; 67(2): 444 - 452. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kajimura, K. Aida, and C. Duan From the Cover: Insulin-like growth factor-binding protein-1 (IGFBP-1) mediates hypoxia-induced embryonic growth and developmental retardation PNAS, January 25, 2005; 102(4): 1240 - 1245. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Cingel-Ristic, J. W. van Neck, J. Frystyk, S. L. S. Drop, and A. Flyvbjerg Administration of Human Insulin-Like Growth Factor-Binding Protein-1 Increases Circulating Levels of Growth Hormone in Mice Endocrinology, September 1, 2004; 145(9): 4401 - 4407. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Chandrashekar, D. Zaczek, and A. Bartke The Consequences of Altered Somatotropic System on Reproduction Biol Reprod, July 1, 2004; 71(1): 17 - 27. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Froment, C. Staub, S. Hembert, C. Pisselet, M. Magistrini, B. Delaleu, D. Seurin, J. E. Levine, L. Johnson, M. Binoux, et al. Reproductive Abnormalities in Human Insulin-Like Growth Factor-Binding Protein-1 Transgenic Male Mice Endocrinology, April 1, 2004; 145(4): 2080 - 2091. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. B. Wheatcroft, M. T. Kearney, A. M. Shah, D. J. Grieve, I. L. Williams, J. P. Miell, and P. A. Crossey Vascular Endothelial Function and Blood Pressure Homeostasis in Mice Overexpressing IGF Binding Protein-1 Diabetes, August 1, 2003; 52(8): 2075 - 2082. |
