| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Unité de Recherche (G.B., D.S., P.G., Y.L.B., S.B.), Institut National de la Santé et de la Recherche Médicale, Unité 515, Croissance, Différenciation et Processus Tumoraux, Hôpital Saint Antoine, 75571 Paris Cedex 12, France; Physiologie de la Reproduction et des Comportements (P.F., P.M.), Unité Mixte de Recherch 6073 Institut National de la Recherche Agronomique-Centre National de la Recherche Scientifique-Université de Tours, 37380 Nouzilly, France; and Service d’Anatomo-Pathologie (M.B.), Hôpital Sainte Anne, 75014 Paris, France
Address all correspondence and requests for reprints to: Sylvie Babajko, Institut National de la Santé et de la Recherche Médicale, Unité 515, Hôpital Saint Antoine, 184 rue du Faubourg St. Antoine, 75571 Paris Cedex 12, France. E-mail: U515{at}st-antoine.inserm.fr.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In man and rodents, IGF-II is implicated mainly in embryonic and fetal growth (3). In rodents, plasma IGF-II concentrations diminish rapidly after birth and continue to decrease into adulthood. Nevertheless, IGF-II expression persists in the central nervous system (CNS), in astroglial structures, and in some sites like the hypothalamus, meninges, brain blood vessels, and the choroids plexus, which is the source of the high levels of IGF-II seen in cerebrospinal fluid (CSF) (3, 4, 5). Studies of neuronal and glial cells in culture have demonstrated the effects of IGFs on cell proliferation and survival (6), and knock-out experiments, particularly involving the IGF-II gene, have confirmed the role of IGFs in CNS development (7, 8)
In all body fluids, IGFs bind noncovalently to high-affinity binding proteins, the IGF binding proteins (IGFBPs). These constitute a family of six molecular species (9) that modulate IGF action, at times potentiating it but more frequently inhibiting it (10). Like the IGFs, IGFBPs are synthesized mainly in the liver, exerting an endocrine action from the bloodstream. Nevertheless, each IGFBP has an individual expression profile in the course of development and in the different organs where they are locally produced and where their action is autocrine/paracrine.
The two major IGFBPs in CSF are IGFBP-2 and IGFBP-6, both of which have preferential affinity for IGF-II (11, 12). IGFBP-6 in the CNS is synthesized in the choroid plexus and by astroglial cells (13). Numerous studies of diverse cell types have shown that IGFBP-6, whether exogenous (administered in recombinant form) or endogenous (obtained by selection of cells overexpressing IGFBP-6), is involved in the arrest of proliferation, an increase in apoptosis, and diminished tumorigenic potency (14, 15, 16). The affinity of IGFBP-6 for IGF-II is 10–100 times that of the other IGFBPs and about 100 times that of the type 1 IGF receptor for IGF-II (11). Therefore, IGFBP-6 can inhibit the effects of IGF-II by sequestration (14).
In view of the roles of IGF-II and IGFBP-6 in the CNS, we considered that transgenic mice overexpressing IGFBP-6 would constitute a good model to study the effects of IGFBP-6 on tissue maturation in vivo. We elected to target IGFBP-6 expression to the CNS by placing transgene expression under the control of the glial fibrillary acidic protein (GFAP) promoter, which has been used successfully in transgenesis (17, 18) in targeting the products of various genes to the CNS (19). GFAP is found in intermediary filaments and is abundantly expressed by CNS astrocytes from d 16 of embryonic life in mice, reaching a plateau 2 wk after birth (20, 21). It is also expressed in several other cell types, including nonmyelinating Schwann cells, hepatic perisinusoidal stellate cells, and testicular Leydig cells (22, 23, 24).
Therefore, we set out to establish lines of transgenic mice overexpressing IGFBP-6 in the CNS and gonads and to explore their phenotypes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
).
|
) and then purified by agarose gel electrophoresis and chromatography on Elutip-d columns (Schleicher & Schuell, Dassel, Germany). The purified insert was diluted in 10 mM Tris-HCl (pH 8) and 0.1 mM EDTA microinjection buffer. Microinjection was carried out as previously described (27) into fertilized oocytes of C57BL6/DBA2 F2 hybrid mice, which were pretreated with 5 U pregnant mare’s serum gonadotropin and then 5 U human chorionic gonadotropin 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 lab chow and maintained in a regulated light cycle (12 h light, 12 h darkness). F1 transgenic mice were mated with B6CBA mice before homozygous transgenic mice were generated, and B6CBA mice were used as wild types.
Blood samples were taken by intraorbital puncture in adults and after decapitation of younger animals, and the samples were collected in 0.13 M EDTA. Plasma samples were centrifuged and then divided into aliquots and stored at –20 C.
Animals were either killed by cervical dislocation and the organs immediately frozen in liquid nitrogen and stored at –70 C for RNA extraction or fixed with 10% formol for histological analysis. These protocols received the approval of the French Ministry of Research and Technology (No. 3299B).
Astrocyte primary culture
Brains of 1- to 3-d-old mice were rapidly excised and crushed in MEM (Invitrogen Life Technologies, Cergy Pontoise, France). Cells were filtered at 0.75 µm and then incubated in a humidified incubator at 37 C, with 5% CO2 atmosphere, for 3 h to allow fibroblasts to adhere to the plate. Astrocytes in the supernatant were transferred to fresh dishes and cultured for 72 h before being harvested. They were then grown in MEM supplemented with 10% heat-inactivated fetal calf serum in the presence of 100 IU/ml penicillin-streptomycin, 1 µg/ml amphotericin B, amino acids (double concentration), and 0.2 mM ascorbate.
Extraction of genomic DNA and Southern blot analysis
Tail fragments were cut at 3 wk 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% sodium dodecyl sulfate, 5 mM dithiothreitol, and 0.5 mM spermidine at 56 C. High molecular weight DNA was isolated by extraction with phenol/chloroform and precipitated in 100% ethanol (28).
Ten micrograms of 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 (NEN Life Science Products, Boston, MA) for Southern blot analysis (29). All experiments were performed in triplicate.
Isolation of RNA, Northern blotting, and RT-PCR analysis
Total RNA was extracted from frozen tissue using the standard CsCl/guanidine isothiocyanate method (30). Thirty micrograms of total RNA were loaded onto 1.2% agarose/2.2 M formaldehyde gels for electrophoresis, then transferred and covalently bound to Hybond-N nylon membranes (Amersham Biosciences, Saclay, France). After prehybridization, the blots were hybridized for 24 h at 50 C to 3 x 106 cpm/ml hIGFBP-6 cDNA probe (Multiprime DNA Labeling System, Amersham).
RT-PCR was carried out on 1 µg total RNA using the SuperScript TM One-Step RT-PCR kit and Platinium Taq (Invitrogen Life Technologies). The 235-bp GFAP fragment was amplified after 20 cycles of PCR using 5'-AGAACAACCTGGCTGCGTAT as forward primer and 5'-GCCTCGTATTGAGTGCG-AAT as reverse primer. ImageQuant software [Molecular Dynamics (Amersham Biosciences)] was used for densitometric quantification, with GFAP expression standardized against that of glyceraldehyde-3-phosphate dehydrogenase as control. All experiments were performed in triplicate.
Western blot analysis
Conditioned media were desalted on Sephadex G25 columns, lyophilized, and analyzed by Western immunoblotting as previously described (31). Briefly, 1 ml equivalent of each sample (media conditioned by 106 cells over 48 h) or 100 µg mouse serum proteins were subjected to 11% SDS-PAGE under nonreducing conditions. Proteins were electrotransferred onto nitrocellulose membranes, which were blocked with 5% milk and incubated at 37 C for 1 h with either anti-hIGFBP-6 or anti-IGFBP-1 polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:600 and 1:250 dilutions, respectively. The nitrocellulose membranes were rinsed and then incubated for 45 min with goat polyclonal antirabbit IgG antibody coupled to horseradish peroxidase (Sigma Chemical Co., St. Louis, MO) at 1:10000 dilution. Horseradish peroxidase oxidation of luminol (ECL Western blotting detection system, Amersham Pharmacia Biotech) yields chemiluminescence, from which the antibody complexes are visualized. Loading and transfer homogeneity were checked by red Ponceau staining of membranes.
Cerebella were homogenized in 10 vol 50 mM Tris-HCl (pH 7.6) and 1% Triton X-100. Samples were then sonicated for 10 sec and centrifuged at 14,000 x g for 15 min at 4 C, and supernatants were collected. Forty micrograms of proteins were subjected to 10% SDS-PAGE and electrotransferred to nitrocellulose membranes. GFAP was revealed, as described earlier, using a specific goat polyclonal antibody (Santa Cruz Biotechnology). Same membranes were also incubated with mouse monoclonal antibody directed against actin (Sigma) after a first wash with 50 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, and 0.8% mercapto-2-ethanol for 45 min at 55 C and a second wash with 0.05% Tween in PBS for 30 min at room temperature.
Enzymatic deglycosylation of IGFBP-6
Mouse serum proteins (100 µg) were taken up in 50 µl of 0.02 M sodium phosphate (pH 6.6). Samples were incubated at 37 C for 20 h with either 20 mU neuramidase (Roche Diagnostics, Penzberg, Germany) or 20 mU neuramidase and 2 mU of O-glycosidase (Roche). After treatment, IGFBP-6 was revealed by Western blotting as described earlier.
Hormone assays
Circulating LH and FSH concentrations were measured by RIA in 6-month-old mice. For LH, samples were incubated overnight at 4 C with 125I rat LH (20,000 cpm/tube) and rabbit polyclonal antiserum raised against rat LH (1:75,000). Volumes were adjusted to a final volume of 500 µl using a solution of 0.03 M NaH2PO4, 3.72 g/liter EDTA, 500 µl/liter Tween 20, 200 mg/liter protamine sulfate, and 200 mg/liter azide (pH 7.4). Thereafter, samples were incubated overnight at 4 C with sheep antiserum raised against rabbit IgG (6 µl/tube) and polyethylene glycol 6000 (0.06 g/tube) in PBS (2 ml/tube). They were then centrifuged, and radioactivity was counted in the pellet. LH concentrations were calculated on the basis of a range standard between 5 
g and 5 ng mouse LH. The sensitivity of the LH RIA was 20 g/tube. FSH concentrations were measured using the rat 125I FSH assay system (Amersham Biosciences). Briefly, 100-µl plasma samples were incubated for 24 h at room temperature with rat 125 FSH and goat polyclonal antiserum. Samples were then incubated with donkey antisheep serum and centrifuged, and radioactivity was counted in the pellet.
Circulating IGF concentrations were measured in 15-d- and 1- and 3-month-old mice using the rat IGF-I RIA kit from Diagnostic Systems Laboratories (Webster, TX). Briefly, 25-µl plasma samples were extracted using acid-ethanol to separate IGF-I from IGFBPs. Extracted samples were incubated overnight at room temperature with rat 125I-IGF and goat antirat IGF-I serum. Samples were then incubated for 20 min at room temperature with donkey antigoat 
globulin and polyethylene glycerol as carrier. After centrifugation, radioactivity was counted in the pellet.
Histological analysis
Brains and ovaries were fixed in 10% formol for 24 h and overnight, respectively, and then embedded in paraffin, after which, they were sectioned at a thickness of 4 µm and stained with hematoxylin and eosin. GFAP was identified by incubating the slices first with 2 µg/ml goat polyclonal antibody directed against human GFAP, but which cross-reacts with mouse GFAP, and then with rabbit polyclonal antigoat biotin-conjugated antibody (Santa Cruz Biotechnology), and GFAP-expressing cells were revealed using avidin-biotin/peroxidase with diaminobenzidine.
The sizes of 10 brains and cerebella from each group of animals (wild type, homozygous 16 and homozygous 17) were measured with calipers.
Statistical analysis
All data are presented as means ± SEM. Student’s t test and, in the case of heterogeneity of variance, the Mann-Whitney U test were used to compare means between two groups. For multiple comparisons of means, ANOVA was used, followed by the Newman-Keuls test, or Kruskal-Wallis ANOVA, as appropriate. P < 0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
).
hIGFBP-6, migrating at 32 kDa, was identified by Western blotting in media conditioned by astrocytes (obtained from 1- to 3-d-old mice) in primary culture (Fig. 1C).
IGFBP-6 expression in transgenic mice
GFAP is found in intermediary filaments where it is strongly expressed by type 2 astrocytes (17, 18). Northern blotting was used to measure IGFBP-6 mRNA in the brain and rachidian bulb of F1 progeny of founders. The messenger was found in both structures, particularly in the rachidian bulb, as would be expected on the basis of the promoter used, but not in the brains of control littermates (Fig. 2A).
|
, B and C). Northern blot analyses revealed that IGFBP-6 expression in transgenic mouse ovaries was slightly above that of wild-type animals. Moreover, IGFBP-6 expression was observed in the testes of transgenic mice (in smaller amounts than in brain) but not in those of wild-type animals, and its expression clearly increased with age (Fig. 2C). IGFBP-6 mRNA was found in the spleen, and traces were detected in the uterus and liver of transgenic and wild-type animals. There was also evidence of transgene expression in the lungs of transgenic mice, particularly in line 16. IGFBP-6 mRNA was not detectable by Northern blotting in the kidneys (Fig. 2B).
Western blot analysis of the plasma of transgenic animals revealed IGFBP-6 only between the ages of 3 and 15 d; the protein was no longer detectable at 30 d (Fig. 3A). Enzymatic deglycosylation of serum proteins showed that the hIGFBP-6 overexpressed in serum was mostly O-glycosylated (Fig. 3A).
|
).
Plasma IGF-I levels were measured at various ages to characterize the IGF axis in IGFBP-6 transgenic mice. In 15-d-old transgenic mice, plasma IGF levels were decreased (142.6 ± 9.3 ng/ml, n = 20, and 124.3 ± 8.7 ng/ml, n = 20) for lines 16 and 17, respectively, compared with wild types of the same age (199.6 ± 15.6 ng/ml, n = 20, P < 0.001; Fig. 3B). However, there were no significant differences between plasma IGF levels of transgenic and wild-type mice at 1 and 3 months of age.
Weight gain in transgenic animals
Growth retardation was detectable between birth and 1 month of age in transgenic mice (Fig. 4). Mean body weights at birth were 1.38 ± 0.2 g (P < 0.015, n = 59) for line 16 and 1.34 ± 0.17 g (P < 0.0001, n = 60) for line 17, as opposed to 1.46 ± 0.13 g (n = 50) for wild types (Fig. 4A). Transgenic 30-d-old females were also significantly smaller (13 ± 2 g, P < 0.0001, n = 76) than wild types (16 ± 2 g, n = 53; Fig. 4B), as were transgenic males (14 ± 2 g, P < 0.0001, n = 70 vs. 16 ± 2 g, n = 46; Fig. 4C). However, catch-up was total in adult males and females of line 16 from 3 months of age (Fig. 4, B and C), but minor growth restriction persisted in the adult females of line 17.
|
Anomalies of the cerebellum were found in transgenics, where cerebellar folia were thinner and size and weight were homogeneously reduced by about 25% and 35%, respectively (Fig. 5). Analysis of the white matter of cerebellum slices revealed smaller numbers of astrocytes expressing less GFAP than those from wild-type tissue (Fig. 6A). A 50% reduction in cerebellum GFAP mRNA was estimated from densitometric quantification of RT-PCR in both transgenic mouse lines compared with wild-type mice (Fig. 6B). GFAP protein levels were also strongly reduced in transgenic mice, with decreases of 40% and 75% for lines 16 and 17, respectively (Fig. 6C).
|
|
). Unlike wild-type animals, 5–20% of the transgenic females proved to be sterile (over the total series of experiments, 14 of 110 females that mated with a wild-type male failed to become pregnant after 2 months). A further 30 females began to gain weight but then lost weight before giving birth, indicating that some fetuses had been aborted. Among the remainder, litters of young adult transgenic females were small (five to six pups compared with eight to nine pups for wild types crossed with transgenic males, P < 0.0001), and older transgenic females (aged 6 and 9 months) had even smaller litters of three to four pups (Table 1). On average, one pup per litter died within a month of birth. Litters sired by young adult transgenic males were in the normal range, with eight to nine pups (Table 1). Interestingly, when 12-month-old transgenic males were used as sires, three of 12 had become sterile (no impregnation after 2 months with their mates). In the remainder, fertilization of their mates was delayed by a month, but the number of pups per litter was the same as that obtained from wild-type crosses (data not shown).
|
). However, the ovaries of transgenics were smaller (7.21 ± 0.27 mg, P < 0.01 for line 16, and 4.60 ± 0.35 mg, P < 0.0001 for line 17, n = 8) than those of wild types (8.94 ± 0.50 mg, n = 8). Moreover, there were less corpora lutea (17.3 ± 2.9, P < 0.01 for line 17 and 9.5 ± 0.6, P < 0.0001, for line 16 vs. 24.0 ± 0.4 for wild type; Fig. 7B), suggesting some alteration of ovulation.
|
), whereas FSH levels were similar, around 8 ng/ml (Fig. 8B). LH levels in wild-type and transgenic males were the same, around 1.9 ± 0.1 ng/ml.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Two founder mice were obtained, which generated lines of homozygous transgenic progeny producing the expected size of transgene and overexpressing hIGFBP-6 in the CNS as intended. Most, if not all, of the IGFBP-6 was apparently secreted because none was detected by Western blotting in brain protein extracts (data not shown), whereas it was evident in media conditioned by astrocytes in primary culture. To our knowledge, this is the first model of transgenic mouse overexpressing IGFBP-6 to have been reported to date.
We investigated the effects of this IGFBP-6 overexpression in the CNS on cell differentiation. Morphological abnormalities were not apparent in the choroid plexus, hypothalamus, or cortex, or in total cerebral white matter, the normal site of GFAP synthesis. This was possibly because transgene expression was weak during the early stages of brain development. However, in transgenic animals, cerebellum size and weight were uniformly reduced by about 25% and 35%, respectively, with thinner cerebellar lamellae. Immunohistochemistry revealed a smaller number of differentiated GFAP-expressing cells in transgenic mice. Moreover, the proliferation rate (studied by 3H thymidine incorporation) of astrocytes from newborn transgenic mice in primary culture was slower than that of astrocytes from wild-type mice (data not shown). These observations are concordant with results of studies demonstrating the importance of the IGF axis in brain development, especially that of the cerebellum and cortex (35, 36, 37). In these studies, cerebellum weight was increased by 90% in transgenic mice overexpressing IGF-I/-II compared with littermate controls, and among the brain regions examined, the cerebellum exhibited the greatest increase in size. Moreover, IGF-I was shown to influence the growth of Purkinje cells and possibly of other cell types in the cerebellum (36). Transgenic mice overexpressing IGF-I in the CNS also presented considerably larger numbers of cells in the granular layer because IGF-I protects them from apoptosis (37). It has been suggested that the IGFBP-6/IGF-II complex may play a particular role in the trophic maintenance of neurons involved in the coordination of sensorimotor function in the cerebellum (38). All these findings confirm the pivotal role of the IGF system in the cerebellum, where the action of IGFs is trophic and antiapoptotic in both neuronal and glial cells. IGF-II has 10–100 times stronger affinity for IGFBP-6 than for the type 1 IGF receptor (11). IGFBP-6 is thus capable of inhibiting the mitogenic effects of IGF-II on astrocytes by sequestration. Nevertheless, because IGFBPs are also known to influence cell survival independently of their binding to IGFs (9, 39), the question of IGFBP-6 mechanism of action could be raised here. IGF-independent action of IGFBP-6 has been suggested in human neuroblastoma cells (14) and in the survival of oligodendrocyte precursor cells (40). Such effects of IGFBP-6 on astrocyte survival could, therefore, not be excluded, but in our in vivo study, it was not possible to distinguish IGF-independent from IGF-dependent effects. On the basis of phenotypes of other models of IGF and IGFBP transgenic animals, a greater influence of sequestration by IGFBP-6 in our mice would seem more probable.
Another consequence of IGFBP-6 overexpression was a significant weight deficit in newborn to 30-d-old transgenic mice, which was attenuated in older animals. Small size is characteristic of most transgenic mice overexpressing one of the six IGFBPs in the bloodstream (41) and may be explained by sequestration of circulating IGFs. In our model, IGFBP-6 is expressed in the brain during the last days of fetal life because it is under the control of the GFAP promoter (20, 21). During the perinatal period, it could cross the blood-brain barrier, which becomes fully functional only a month postnatally. IGFBP-6 was clearly detectable in the sera of 3- to 15-d-old transgenic mice and had disappeared at the age of 1 month. The absence of serum IGFBP-6 in adult animals was compatible with the absence of IGFBP-6 mRNA detected in the liver, the primary source of circulating IGFBPs. In the serum, hIGFBP-6 was mostly O-glycosylated. O-glycosylation delays the clearance of IGFBP-6 from the circulation (42) and maintains IGFBP-6 in a soluble form by inhibiting binding of IGFBP-6 to glycosaminoglycans and cell membranes (43). Up to 1 month of age, IGFBP-6 would exert its effects by sequestering IGFs and inhibiting their endocrine effects on growth (44). Moreover, circulating levels of IGF-I were depressed, whereas IGFBP-1 levels were still high in 15-d-old mice of both transgenic lines compared with the wild-type line. All these events contribute to the reduced IGF effect on growth and explain growth retardation during this period. Nevertheless, it cannot be excluded that this transient growth retardation could also be due to a defect in nutrition during fetal life as suggested by the slight but significant lower body weight at birth (45).
The most striking phenotype among our transgenic animals was reproductive deficiency. In IGFBP-6 transgenic males, sterility increased with age to approximately 20% of 9- to 12-month-old males, whereas none of the wild-type males used in the study were sterile. IGFBP-6 mRNA was detectable by Northern blotting in the testes of transgenic mice and also increased with age. The increased sterility could, therefore, result from local IGFBP-6 transgene expression in the testis (46).
Matings between wild-type males and over 100 homozygous 3-month-old females revealed 5–20% sterility after 2 months with the males (depending on the time of year of the series of experiments). Litter size for these young adults averaged around six pups, as opposed to nine to 10 pups for wild types. These abnormalities were accentuated in older transgenic females (aged between 6 and 9 months), with only around three pups per litter, suggesting precocious aging of reproductive function. Analysis of transgene expression in different organs indicated weak IGFBP-6 expression in the ovaries, which was slightly above that in wild types, suggesting possible alteration by IGFBP-6 of IGF action on folliculogenesis (47, 48). Nevertheless, unlike reproductive deficiency, transgene expression in the ovaries did not increase with age. The cause of the reproductive deficiency appeared to be an alteration of ovulation (reduced corpora lutea) and a dramatic decrease in plasma LH concentrations. These data suggest some hypothalamo-pituitary disorder resulting from hIGFBP-6 overexpression in the brain. IGF-II has been shown to be involved in gonadotropin release in the GTI-7 hypothalamic GnRH neuron cell line (49), which expresses IGFBPs (IGFBP-2 to -6), IGFs (IGF-I and -II), and the type 1 IGF receptor, showing that the IGF system plays an autocrine regulatory role in the secretory activities of GnRH neurons (50). The link between IGF system and GnRH expression was recently confirmed in vivo (51).
Unlike other models of transgenic mouse described to date, such as IGF-I–/– (52), GH receptor–/– (53), or IGFBP-1 (54), in which the IGF system is affected and reproductive deficiency is associated with growth restriction, in our model of the IGFBP-6 transgenic mouse, small size and reproductive deficiency appeared to be independent of each other. In adult mice, disturbances of the IGF system in the CNS clearly had severe repercussions on fertility without affecting levels of circulating IGFs or body size. Our results also show that females are far more sensitive to these disturbances than males, with markedly reduced plasma LH levels, as has been reported for NIRKO mice (46).
Because IGFBP-6 is secreted in CSF (11, 12), it would be interesting to investigate its expression in the brain in animals with reproductive defects to see whether a direct relationship exists between IGFBP-6 expression and sterility.
In conclusion, our findings indicate that this new model of transgenic mouse may prove to be a useful tool in elucidating the role in vivo of the IGF system in the CNS and specifically the part played by IGFBP-6 in the maturation of the cerebellum and pituitary-hypothalamic physiology.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: CNS, Central nervous system; CSF, cerebrospinal fluid; GFAP, glial fibrillary acidic protein; IGFBP, IGF binding protein.
Received September 9, 2003.
Accepted for publication January 21, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  X. Wang, L. Lu, Y. Li, M. Li, C. Chen, Q. Feng, C. Zhang, and C. Duan Molecular and functional characterization of two distinct IGF binding protein-6 genes in zebrafish Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2009; 296(5): R1348 - R1357. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. P. Sperandeo, P. Annunziata, A. Bozzato, P. Piccolo, L. Maiuri, M. D'Armiento, A. Ballabio, G. Corso, G. Andria, G. Borsani, et al. Slc7a7 disruption causes fetal growth retardation by downregulating Igf1 in the mouse model of lysinuric protein intolerance Am J Physiol Cell Physiol, July 1, 2007; 293(1): C191 - C198. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. B. Wheatcroft, M. T. Kearney, A. M. Shah, V. A. Ezzat, J. R. Miell, M. Modo, S. C.R. Williams, W. P. Cawthorn, G. Medina-Gomez, A. Vidal-Puig, et al. IGF-Binding Protein-2 Protects Against the Development of Obesity and Insulin Resistance Diabetes, February 1, 2007; 56(2): 285 - 294. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Ben Lagha, D. Seurin, Y. Le Bouc, M. Binoux, A. Berdal, P. Menuelle, and S. Babajko Insulin-Like Growth Factor Binding Protein (IGFBP-1) Involvement in Intrauterine Growth Retardation: Study on IGFBP-1 Overexpressing Transgenic Mice Endocrinology, October 1, 2006; 147(10): 4730 - 4737. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. H. Larsen, A. G. DaSilva, K. Conant, and V. W. Yong Myelin formation during development of the CNS is delayed in matrix metalloproteinase-9 and -12 null mice. J. Neurosci., February 22, 2006; 26(8): 2207 - 2214. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. C. Russo, P. D. Gluckman, E. L. Feldman, and G. A. Werther The Insulin-Like Growth Factor System and Its Pleiotropic Functions in Brain Endocr. Rev., December 1, 2005; 26(7): 916 - 943. [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]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
