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Am J Physiol Endocrinol Metab 291: E333-E339, 2006. First published February 28, 2006; doi:10.1152/ajpendo.00045.2006
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Effect of pulsatile growth hormone administration to the growth-restricted fetal sheep on somatotrophic axis gene expression in fetal and placental tissues

F. H. Bloomfield, P. L. van Zijl, M. K. Bauer, H. H. Phua, and J. E. Harding

The Liggins Institute, University of Auckland, Auckland, New Zealand

Submitted 30 January 2006 ; accepted in final form 27 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have previously reported (Bauer MK, Breier BH, Bloomfield FH, Jensen EC, Gluckman PD, and Harding JE. J Endocrinol 177: 83–92, 2003) that a chronic pulsatile infusion of growth hormone (GH) to intrauterine growth-restricted (IUGR) ovine fetuses increased fetal circulating IGF-I levels without increasing fetal growth. We hypothesized a cortisol-induced upregulation of fetal hepatic GH receptor (GH-R) mRNA levels, secondary increases in IGF-I mRNA levels, and circulating IGF-I levels, but a downregulation of the type I IGF receptor (IGF-IR) as an explanation. We, therefore, measured mRNA levels of genes of the somatotrophic axis by real-time RT-PCR in fetal and placental tissues of fetuses with IUGR (induced by uteroplacental embolization from 110- to 116-days gestation) that received either a pulsatile infusion of GH (total dose 3.5 mg/day) or vehicle from 117–126 days and in control fetuses (n = 5 per group). Tissues were collected at 127 days (term, 145 days). Fetal cortisol concentrations were significantly increased in IUGR fetuses. However, in liver, GH-R, but not IGF-I or IGF-IR, mRNA levels were decreased in both IUGR groups. In contrast, in placenta, GH-R, IGF-I, and IGF-IR expression were increased in IUGR vehicle-infused fetuses. GH infusion further increased placental GH-R and IGF-IR, but abolished the increase in IGF-I mRNA levels. GH infusion reduced IGF-I expression in muscle and increased GH-R but decreased IGF-IR expression in kidney. IUGR increased hepatic IGF-binding protein (IGFBP)-1 and placental IGFBP-2 and -3 mRNA levels with no further effect of GH infusion. In conclusion, the modest increases in circulating cortisol concentrations in IUGR fetuses did not increase hepatic GH-R mRNA expression and, therefore, do not explain the increased circulating IGF-I levels that we found with GH infusion, which are likely due to reduced clearance rather than increased production. We demonstrate tissue-specific regulation of the somatotrophic axis in IUGR fetuses and a discontinuity between GH-R and IGF-I gene expression in GH-infused fetuses that is not explained by alterations in phosphorylated STAT5b.

fetal therapy; intrauterine growth restriction

INTRAUTERINE GROWTH RESTRICTION remains a major cause of perinatal morbidity and mortality. Epidemiological evidence in humans, substantiated by research in animals, suggests that smaller size at birth is also associated with an increased risk of adverse, long-term health outcomes (22). However, there is not yet any effective intrauterine treatment for the fetus identified as growth restricted. In attempting to develop such treatments, we have used the sheep as our experimental paradigm and have focused on supplementation of the mother or fetus with hormones of the somatotrophic axis, as the major regulators of growth before and after birth (14).

After birth, the somatotrophic axis regulates growth predominantly via growth hormone (GH) acting on the GH receptors (GH-R) to increase tissue and circulating IGF-I levels. However, before birth, GH has been thought to have only a minor role in regulating fetal growth. Fetal sheep made GH deficient by hypophysectomy are of normal birth weight and have only mildly reduced circulating IGF-I levels (23, 24). Similarly, human infants with congenital GH deficiency show only a small reduction in birth length (10). In contrast, the IGFs are the principle regulators of fetal growth (11), with IGF-I levels in sheep being regulated mainly by nutritional factors (26, 27). In sheep, plasma GH concentrations are 10- to 20-fold higher during fetal life than after birth, yet circulating IGF-I levels are comparatively low (7). This dissociation between GH and IGF-I levels before birth may be partly explained by the low levels of GH-R in the ovine fetal liver, which are only ~30% of adult levels (16). It has been shown in fetal sheep that hepatic GH-R mRNA levels are regulated by cortisol, increasing toward term in parallel with the prepartum cortisol surge (19). Abolition of this surge by fetal adrenalectomy prevents the rise in GH-R, and elevating fetal plasma cortisol levels by exogenous infusion early in gestation results in a premature rise in hepatic GH-R levels (19). After birth, both GH-R and circulating IGF-I levels increase rapidly in lambs, whereas circulating GH levels fall to adult levels (4). The ovine GH-R gene has two transcripts, due to alternate splice variants of an untranslated exon (2). The increases in hepatic GH-R mRNA levels in fetuses approaching term and following glucocorticoid infusion, and also in postnatal lambs, are largely due to an increase in transcription of exon 1A-derived GH-R mRNA (2, 18), a form that is found in adult tissues and that contains putative half-site glucocorticoid response elements (25).

Our laboratory has previously reported that a pulsatile infusion of GH to growth-restricted fetal sheep for 10 days from 117 days gestation (term, 145 days) resulted in an unexpected increase in circulating levels of IGF-I, but, paradoxically, no increase in fetal growth (3). In these sheep, growth restriction was induced by placental embolization, which has previously been shown to elevate fetal circulating cortisol concentrations (9). We, therefore, hypothesized that, in our study, embolization may have caused an elevation in fetal cortisol concentrations, which, in turn, was responsible for an upregulation of hepatic exon 1A-derived GH-R mRNA levels. As the liver is the main site of production of circulating IGF-I, the exogenous GH would then act via the upregulated hepatic GH-R to increase production of IGF-I. To explain why this increased IGF-I did not increase fetal growth, we further hypothesized a compensatory downregulation of the IGF type I receptor (IGF-IR), which may have occurred to prevent the fetus growing faster than the available nutrient supply could support. This present study was designed to test these hypotheses by measuring mRNA levels of GH-R, IGF-I, and IGF-IR in fetal and placental tissues.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Animal experiments were approved by the Animal Ethics Committee of the University of Auckland and have been reported previously in detail (3). In brief, 15 ewes carrying singleton fetuses underwent surgery at 106 days of gestation (term = 145 days) for implantation of chronic indwelling vascular and amniotic catheters. After surgery, ewes were housed in individual metabolic cages with free access to water and concentrates. Ewes were randomly assigned to three treatment groups: controls and growth-restricted fetuses treated with either saline or GH (n = 5 per group). Growth restriction was induced by embolization of the placental vascular bed from 110–116 days gestation, and growth-restricted fetuses were then infused in a pulsatile manner with either saline or GH (total of 3.5 mg/day, composed of a continuous infusion delivering 2.5 mg/day and 30 superimposed pulses of 330 µl each delivering 1 mg/day) for 10 days. Control fetuses were unembolized and untreated. At 127 days gestation, ewes were killed with an overdose of pentobarbitone, and the uterus and contents were dissected, weighed, and measured. Tissues were snap frozen in liquid nitrogen and stored at –80°C until analysis.

Cortisol measurements. A new method was developed for measuring cortisol by mass spectrometry, based on published data (30). The HPLC mass spectrometer system consists of a Surveyor MS pump and Surveyor auto sampler followed by an Ion Max atmospheric pressure chemical ionization source on a Finnigan TSQ Quantum Ultra AM triple quadrupole mass spectrometer, all controlled by Finnigan Xcaliber software (Thermo Electron, Waltham, MA). Plasma (80 µl) was mixed with 50 µl of methanol containing 10 ng/ml [2H2]cortisol as an internal standard. The mixture was filtered through a Nanosep filtration tube with a 3,000 molecular weight (MW) cutoff (Pall Gelman Sciences, Ann Arbor, MI), and 25 µl of filtrate were injected directly onto the HPLC column. The chromatography conditions consisted of a Gemini column (5 µm, C18, 110 Å, 150 x 2 mm, Phenomenex, Torrance, CA) protected by a security guard cartridge (Gemini C18, 4 x 2 mm, Phenomenex), with a mobile phase consisting of 65% methanol, 10 mM N-methyl-morpholine, 0.2 mM formic acid, flowing at 600 µl/min and with a column temperature of 30°C.

The mass spectrometry conditions consisted of atmospheric pressure chemical ionization in negative mode with a discharge current of 5 A, vaporizer temperature of 425°C, a sheath gas flow of 35 psi, an auxiliary gas flow of 7 psi, a capillary temperature of 250°C, and argon at 1.2 mTorr as the collision gas with a voltage of 17 V. Cortisol is present mainly as M + formate, giving a parent ion of 407.25 MW. The selective reaction monitoring transition was therefore 407.25 -> 331.1 MW (internal standard 409.25 -> 333.1 MW). The standard curve was linear from 6 to 1,000 pg of cortisol loaded onto the column when spill from cortisol into the internal standard channel was corrected for (3% of the cortisol area).

Recovery of cortisol from plasma using this method was tested by spiking fetal plasma with 1 and 5 ng/ml cortisol in quadruplicate and determining cortisol concentrations in all samples, including quadruplicate blank plasma. Recoveries were 108 and 103%, respectively. The inter- and intra-assay coefficients of variation for the assay at the concentrations of cortisol seen in this study were 7.5 and 5.4%, respectively. We also tested samples with different cortisol concentrations. With cortisol concentrations around 35 ng/ml, the inter- and intra-assay coefficients of variation were 2.4 and 1.8%, respectively, and with cortisol concentrations around 1 ng/ml, inter- and intra-assay coefficients of variation were 17.2 and 12.9%, respectively. Cortisol concentrations obtained in both fetal and maternal ovine plasma using this method are similar to those obtained in our laboratory using an in-house radioimmunoassay.

Real-time relative quantitative RT-PCR. Total RNA was extracted by the TRIzol method (GIBCO-BRL, Life Technologies, Auckland, New Zealand). The final concentration of RNA was quantified using spectrophotometric 260-nm optical density (OD260) measurements, and purity was assessed by OD260/OD280 ratio, with values >1.9 being of acceptable purity. RNA electrophoresis on 1.2% formaldehyde agarose gels followed by ethidium bromide staining was used to verify RNA integrity and also to confirm that the genes being investigated were present and of predicted size. Expression levels were quantitated using a one-step PCR reaction, as described previously (31). Real-time PCR efficiencies (E) were calculated from the slopes of the standard curves for each target gene (E = 10–1/slope). Samples from the control group were selected as a calibrator. Gene expression in the vehicle and GH-treated groups were expressed relative to the calibrator and as a ratio to 18S rRNA using the formula (28):

Formula
where Etarget is the real-time PCR amplification efficiency of target gene transcript; E18S is the real-time PCR amplification efficiency of 18S; {Delta}CPtarget (control – treated) and {Delta}CP18S (control – treated) are the cycle threshold differences between the calibrator (the control group) and treated group (calculated separately for the vehicle and GH-treated groups) for the target gene and 18S rRNA, respectively.

Data for real-time PCR are, therefore, expressed as relative expression ratios to the control group with 95% confidence intervals. When confidence intervals between groups do not overlap, the groups are significantly different from each other at the 5% level.

Oligonucleotide primers and probe design. Gene-specific primers and probes for target genes were designed using the ABI Primer Express software (Applied Biosystems, Foster City, CA) and were synthesized by Invitrogen (Auckland, NZ) and Applied Biosystems, respectively. Each probe was dual labeled at the 5' end with fluorescein [for GH-R, IGF-I, IGF-IR, IGF-binding protein (IGFBP)-1, and IGFBP-2] or VIC (for ribosomal 18S RNA) and at the 3' end with a minor groove-binder nonfluorescent quencher. Primer and probe sequences for IGF-I were located within exon 3 (GenBank accession number M31724; amplicon 91 bp; forward primer: bp 241–260; reverse primer: bp 314–331; probe: bp 262–277). Primer sequences for IGF-IR were specific for the beta-subunit (accession number AY162434; amplicon 64 bp; forward primer: bp 73–97; reverse primer: bp 118–136; probe: bp 100–115). Primer sequences for GH-R were located within exon 1A (accession number M82912; amplicon 59 bp; forward primer: bp 119–140; reverse primer: bp 159–177; probe: bp 142–157). The probes and primers for IGFBP-1 and -2 were as described previously (31). The probe and primer for ribosomal 18S RNA (18S rRNA) were obtained from Applied Biosystems (Assay-on-Demand, Applied Biosystems).

Western blots and immunoprecipitation. Protein was extracted from tissues, quantified, separated electrophoretically, and transferred to nitrocellulose membranes, and nonspecific binding was blocked as described previously (31). Blots were then incubated with a polyclonal rabbit anti-human IGF-I antibody (Abcam, Cambridge, UK) at 1:1,000 dilution for 1 h at room temperature. Following PBS-0.1% Tween-20 washes, blots were incubated with a mouse monoclonal anti-rabbit horseradish peroxidase conjugate (Sigma Chemical) at a 1:10,000 dilution in blocking solution for 1 h at room temperature. Specific protein bands were detected, captured, and analyzed as described previously (31). Membranes were then stripped and processed for detection of beta-actin [primary mouse anti-human monoclonal antibody, dilution 1:2,500 (Abcam), secondary antibody polyclonal rabbit anti-mouse horseradish peroxidase conjugate, dilution 1:10,000 (Abcam)]. A control lane consisting of a standard stock sample of fetal muscle was run on each gel to confirm equal loading and transfer across gels. Images were analyzed using Scion Image (Scion, Frederick, MD). All signals are expressed as optical densities relative to the signal for beta-actin.

Immunoprecipitation for phosphorylated STAT5b. Tissues were homogenized in lysis buffer containing phosphatase inhibitors [50 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EGTA, 2 mM EDTA, 0.2% Triton X-100, 0.3% Nonidet P-40, 25 mM NaF, 25 mM beta-glycerolphosphate, 100 µM sodium orthovanadate, and Complete Mini EDTA free protease inhibitor (Roche)]. After centrifugation (8,000 g) at 4°C, the lysates were incubated with 50 µl (50% slurry) of protein G-Sepharose (Sigma), and rocked at 4°C for 1 h. After further centrifugation, the supernatants were transferred to fresh tubes, and the protein concentration was determined as above. Protein (500 µg) was incubated with mouse anti-STAT5b antibody (10 µg, Zymed Laboratories) for 2 h at 4°C on a rocker. Afterwards, immune complexes were collected with protein G-Sepharose for 1 h at 4°C on a rocker. Finally, after extensive washing with lysis buffer and boiling for 5 min in 1 x SDS-PAGE sample buffer, the immunoprecipitates were subjected to SDS-PAGE and analyzed by Western blotting using rabbit anti-phospho-STAT5b antibody (1:500; Zymed Laboratories). Blots were incubated with a mouse anti-rabbit horseradish peroxidase-conjugated secondary antibody (Sigma Chemical), and bands were detected and analyzed as above. Relative or absolute optical densities of bands on Western blots are expressed as mean (SD) and were analyzed by ANOVA (Statview, SAS Institute, Cary, NC) with significance set at the 5% level. Cortisol concentrations at baseline [mean (SD)] were compared by ANOVA. A repeated-measures ANOVA for change of cortisol from baseline was used to compare cortisol concentrations among groups over time. If significant differences at the 5% level were found, the Games Howell post hoc test was used to compare between groups.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Data on fetal growth, placental size, fetal and organ weights, and plasma IGF-I and IGFBP levels have been reported previously (3), and salient findings are outlined in the Introduction. Basic physiological data are also presented in Table 1 for completeness. Fetal circulating cortisol concentrations at baseline were lower in the vehicle-infused group, but this was not statistically significant (control 1.6 (1.3); vehicle 0.6 (0.1); GH 1.1 (0.9) ng/ml; P = 0.2). Change in fetal circulating cortisol concentration from baseline over the period of the study was significantly greater in both embolized groups compared with the control group (P < 0.05; Fig. 1). Maternal circulating cortisol concentrations were not significantly different among groups either at baseline (control 6.2 (6.6); vehicle 11.0 (14.5); GH 13.2 (12.0) ng/ml; P = 0.6) or over the period of the study (Fig. 1).


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  		Table 1. Fetal arterial substrate concentrations before commencing treatment and fetal substrate uptake rates at the end of the 10-day treatment period

 

Figure 1
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  		Fig. 1. Change ({Delta}) in arterial cortisol concentrations from baseline (110 days gestation) in fetuses (A) and ewes (B) throughout the study. {square}, Control animals; {circ}, vehicle-infused animals; bullet, GH-infused animals (n = 5 per group). Values are mean (SD). Solid bar represents the period of embolization. Cortisol concentrations increased significantly in embolized fetuses compared with controls (repeated-measures ANOVA, *P < 0.05).

 
Contrary to our hypothesis, hepatic GH-R mRNA levels were decreased by ~60% in vehicle-infused embolized fetuses compared with control fetuses (P < 0.05; Table 2). In contrast, GH-R mRNA levels were increased twofold in embolized fetuses in the placenta (P < 0.05) and were unchanged in muscle and kidney. Fetal GH infusion of embolized fetuses did not alter the low GH-R mRNA levels in the liver, but further increased the elevated levels in the placenta (P < 0.05 vs. control and vehicle groups, Table 2). GH infusion also increased GH-R mRNA levels fourfold in kidney (P < 0.05 compared with both control and vehicle groups), but did not change the levels in muscle (Table 2).


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  		Table 2. mRNA levels of GH-R, IGF-I, and IGF-IR in fetal liver, muscle, and kidney and in placenta

 
IGF-I mRNA and protein levels in liver and muscle were not significantly different between vehicle-infused embolized and control fetuses (Table 2). In placenta of vehicle-infused fetuses, IGF-I mRNA levels were significantly increased compared with controls, consistent with the upregulated GH-R mRNA levels (Table 2). IGF-I mRNA levels were significantly reduced in kidney of vehicle-infused fetuses compared with controls (Table 2). IGF-I mRNA levels were lowest in GH-infused fetuses in all tissues, but were only significantly lower than controls in muscle and significantly lower than vehicle-infused fetuses in placenta, in which GH-infused fetuses had the highest GH-R mRNA levels (Table 2). Similarly, in the kidney, in which GH-infused fetuses had a fourfold increase in GH-R mRNA levels, IGF-I levels were reduced by more than one-half, although this was not statistically significant (Table 2). Protein levels of IGF-I were not statistically different among groups [IGF-to-beta-actin ratios for liver: control 0.76 (0.33), vehicle 0.58 (0.23), GH 0.77 (0.28); placenta: control 0.49 (0.16), vehicle 0.75 (0.21), GH 0.60 (0.21); muscle: control 0.84 (0.70), vehicle 0.89 (0.51), GH 0.62 (0.22); kidney: control 0.62 (0.19), vehicle 0.64 (0.12), GH 0.57 (0.10)].

To investigate whether the dissociation of GH-R and IGF-I levels in liver and placenta may be due to postreceptor suppression of GH signaling, we performed immunoprecipitation of STAT5b and measured levels of the phosphorylated protein by western blot. There were no differences in levels of phosphorylated STAT5b among groups in liver or placenta [arbitrary densitometric units in liver: control 7,362 (1,034), vehicle 8,947 (1,608), GH 9,090 (433); in placenta: control 8,360 (976), vehicle 9,091 (1,713), GH 8,547 (1,713)].

Hepatic and muscle IGF-IR mRNA levels were not different among groups, but placental levels were increased in both embolized groups compared with control fetuses (Table 2). Renal IGF-IR mRNA levels were significantly reduced with GH infusion compared with control fetuses (Table 2).

These data do not suggest that pulsatile GH infusion of intrauterine growth-restricted (IUGR) fetuses resulted in elevated plasma IGF-I levels secondary to increased tissue production of IGF-I. We, therefore, measured mRNA levels of IGFBP-1, -2, and -3 in liver and placenta to determine whether differences in IGF-I binding capacity or clearance may explain this finding. In liver, IGFBP-1 mRNA levels were significantly increased in vehicle-infused fetuses, but not GH-infused fetuses, compared with controls (Table 3). There were no differences in hepatic mRNA levels for IGFBP-2 or -3 among groups. In placenta, IGFBP-1 levels were not different among groups, but IGFBP-2 mRNA levels were significantly higher in vehicle-infused fetuses, and IGFBP-3 mRNA levels were significantly higher in both vehicle- and GH-infused fetuses compared with control fetuses (Table 3).


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  		Table 3. mRNA levels of IGFBP-1, -2, and -3 in fetal liver and placenta

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have previously shown that a 10-day pulsatile infusion of GH to the growth-restricted fetus increased fetal plasma IGF-I levels but did not increase fetal growth (3). The results of the present study do not support the hypothesis that we proposed to explain these findings, namely, that elevated fetal cortisol levels had resulted in increased hepatic GH-R and hence IGF-I expression, while reduced IGF-IR expression prevented any increase in fetal growth. Although fetal plasma cortisol levels were elevated, fetal hepatic GH-R mRNA levels were reduced rather than increased. Furthermore, there was no direct relationship between GH-R mRNA levels and tissue IGF-I mRNA and protein levels, and IGF-IR mRNA levels were only reduced in the fetal kidney. Thus the explanation for the apparently paradoxical results of our previous study remain uncertain.

Previous studies have demonstrated an upregulation of hepatic GH-R and IGF-I mRNA in fetal sheep as term approached (2, 18, 19). By elegant studies involving both adrenalectomy, which prevented the rise of both cortisol and GH-R mRNA levels, and cortisol replacement to similar levels to those seen in the preparturient period, resulting in a similar increase in hepatic GH-R mRNA levels, Li et al. (18) clearly demonstrated a role for cortisol in the regulation of hepatic exon 1A-derived GH-R expression. The alternate transcript, exon 1B-derived GH-R, showed a more gradual ontogenic increase; this transcript does not contain a glucocorticoid response element in the promotor region (1), unlike the exon 1A-derived transcript, but does contain a putative activated thyroid hormone receptor binding site (1). Cortisol, therefore, may regulate hepatic GH-R mRNA levels directly via the exon 1A promotor and indirectly via the exon 1B promotor, where its effects are mediated by thyroid hormones (8). Thus, although thyroid hormone concentrations are reduced in growth-restricted fetuses (32, 35), this is unlikely to be the explanation for the reduced levels of hepatic GH-R mRNA that we found, as we measured the exon 1A transcript. Unfortunately, we did not have sufficient blood to measure fetal thyroid hormone concentrations in this study.

In the studies by Li et al. (18, 19), hepatic GH-R mRNA levels were only increased when cortisol concentrations were in the order of 30 mg/ml or greater. In our study, although there was a significant increase in circulating cortisol concentrations, levels were still considerably lower than seen with the preparturient cortisol surge and were all <10 ng/ml. It may be that these concentrations were insufficient to result in an increase in hepatic GH-R mRNA expression levels, which were, in fact, significantly decreased in the embolized groups.

In contrast, embolization significantly increased GH-R mRNA levels in the placenta, and GH administration increased these further, demonstrating tissue-specific regulation of the fetal somatotrophic axis, consistent with previous observations in both growth-restricted (6, 31) and normal fetuses (17, 19). We are not aware of any previous reports of GH-R mRNA levels in the placenta following manipulation of fetal growth. Of note is the different relationship between GH-R and IGF-I mRNA levels in the placenta between GH- and vehicle-infused fetuses. In the GH-infused group, GH administration increased GH-R mRNA levels, but without an increase in IGF-I levels. In contrast, in the vehicle-infused, growth-restricted fetuses, placental GH-R mRNA levels and IGF-I mRNA levels were both increased twofold. A similar pattern of elevated GH-R mRNA levels without a change in IGF-I mRNA levels was seen in the kidney of the GH-infused fetuses. An increase in fetal renal GH-R mRNA levels without an increase in IGF-I mRNA levels has also been reported in late-gestation sheep following 30 days of maternal undernutrition in early pregnancy (5). The relationship between GH-R and IGF-I mRNA levels in the growth-restricted fetus, therefore, appears to have been altered with GH administration, leading to a discontinuity between GH-R and IGF-I gene expression.

We investigated whether this dissociation between GH-R and IGF-I expression could be due to altered signaling by STAT5b. STAT5b is a member of the signal transducer and activator of transcription (STAT) family and is phosphorylated by the receptor-associated Janus kinase (JAK2) following binding of GH to its receptor. Once phosphorylated, the STATs translocate to the nucleus, where they activate transcription of target genes after binding to conserved regulatory sequences of the genome (15). Studies in rats have demonstrated that STAT5b mediates GH-induced activation of IGF-I and IGFBP-3 in the liver (34), but there are no data regarding its role in GH signaling in the placenta. Our finding that phosphorylated STAT5b protein levels were not different among groups suggests that perturbation of this pathway cannot explain the dissociation between GH-R and IGF-I expression in the placenta and kidney in our study.

Although plasma IGF-I levels were elevated in GH-treated, growth-restricted fetuses (3), we did not find any evidence that this was due to increased IGF-I production. In addition to elevated circulating IGF-I levels, GH-infused fetuses also had a transient rise in circulating IGFBP-1 levels and a prolonged rise in circulating IGFBP-2 levels, with IGFBP-2 levels still approximately twice those in vehicle-infused fetuses at the end of the 10-day infusion period (3). We, therefore, speculated that the increase in IGF-I levels may have been due to the increased levels of IGFBPs, resulting in reduced clearance of IGF-I. However, mRNA levels of IGFBP-1, -2, and -3 were not increased compared with vehicle-infused fetuses. The relatively small increases in hepatic and placental IGFBP mRNA levels that we report with intrauterine growth restriction in the vehicle-infused fetuses are consistent with alterations in IGFBP expression in guinea pigs in which IUGR was induced by uterine artery ligation (6), although in that study IGFBP-2 mRNA levels were increased in liver and not in placenta, as in our study. We have not investigated possible differences in posttranslational modifications of the IGFBPs, such as phosphorylation, glycosylation, and proteolysis, which are known to affect binding (21), nor have we investigated other IGFBPs such as IGFBP-4, -5, and -6, for which there are relatively few data in the sheep.

Despite IGF-I being a principle regulator of fetal growth, the elevated fetal circulating IGF-I levels that occurred in GH-treated fetuses did not result in increased fetal growth (3). We used indwelling growth catheters around the fetal chest, which provide an accurate and sensitive measure of fetal growth rate (12, 13). Nevertheless, this may not be sensitive enough to detect a subtle alteration in growth rate, if one existed. Alternatively, IGF-I action may have been modulated, either by alterations in binding protein affinity or by alterations in the type 1 receptor. There was no difference between vehicle- and GH-infused growth-restricted fetuses in mRNA levels of IGF-IR in placenta, liver, or muscle, although placental levels were significantly increased in both groups compared with control fetuses. In contrast, in the kidney, which was significantly lighter in GH-infused fetuses than in either control or vehicle-infused fetuses, IGF-IR mRNA levels were significantly reduced, suggesting that reduced action of IGF-I via the type 1 receptor may have been responsible for this reduction in kidney size. This is consistent with the reported major role of IGF-I and the IGF-IR in renal development in rodents (20, 29, 33).

In this paper, we also report a new, rapid, robust, and sensitive mass spectrometric method for the measurement of plasma cortisol concentrations that does not require a time-consuming extraction procedure nor use of radioisotopes. Circulating levels obtained with this method in both fetal and maternal plasma are similar to those obtained with an in-house radioimmunoassay (data not shown) and with those in the literature.

In conclusion, we have shown that the elevated circulating IGF-I concentrations in IUGR ovine fetuses infused with a chronic pulsatile infusion of GH are not due to cortisol-induced increases in GH-R expression and a switch to the mature mode of somatotrophic regulation. Rather, they appear to be due to reduced clearance of IGF-I from the circulation. The mechanism of the reduced clearance is not evident from this study, but it does not appear to be due to increased production of IGFBP-1, -2, or -3. Neither does the failure of the elevated circulating IGF-I concentrations to increase fetal growth appear to be due to reduced expression of the IGF-IR in fetal liver or muscle, although the reduction in kidney weight in GH-infused fetuses may be related to reduced IGF-IR expression in this organ.

We have demonstrated tissue-specific regulation of GH-R in growth-restricted fetuses. Furthermore, placental embolization results in increased placental GH-R mRNA, IGF-I, and IGF-IR expression, suggesting that the placenta may form an important part of the somatotrophic axis before birth, which warrants further investigation. However, GH administration to growth-restricted fetuses results in a discontinuity between GH-R and IGF-I mRNA levels that is not explained by alterations in the STAT5b pathway, leading us to conclude that these data do not support the use of fetal GH infusion as a treatment stratagem for the IUGR fetus.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The University of Auckland Vice Chancellor's Committee and the Health Research Council of New Zealand funded this study.


   ACKNOWLEDGMENTS
 
We acknowledge the technical assistance of Toni Smith.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. Bloomfield, Liggins Institute, Univ. of Auckland, Private Bag 92019, Auckland, New Zealand (e-mail: f.bloomfield{at}auckland.ac.nz)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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  3. Bauer MK, Breier BH, Bloomfield FH, Jensen EC, Gluckman PD, and Harding JE. Chronic pulsatile infusion of growth hormone to growth-restricted fetal sheep increases circulating fetal insulin-like growth factor-I levels but not fetal growth. J Endocrinol 177: 83–92, 2003.[Abstract]
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