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Am J Physiol Regul Integr Comp Physiol 288: R1620-R1627, 2005. First published January 20, 2005; doi:10.1152/ajpregu.00789.2004
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DEVELOPMENTAL PHYSIOLOGY AND PREGNANCY

Uteroplacental restriction in the rat impairs fetal growth in association with alterations in placental growth factors including PTHrP

Mary E. Wlodek,1 Kerryn T. Westcott,1 Rachael O’Dowd,1 Anne Serruto,1 Lesley Wassef,1 Karen M. Moritz,3 and Jane M. Moseley2

1Department of Physiology, University of Melbourne, Melbourne; 2Department of Medicine and St. Vincent’s Institute of Medical Research, University of Melbourne, Fitzroy; and 3Department of Anatomy and Cell Biology, Monash University, Clayton, Victoria, Australia

Submitted 22 November 2004 ; accepted in final form 17 January 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
During pregnancy, parathyroid hormone-related protein (PTHrP) is one of many growth factors that play important roles to promote fetal growth and development, including stimulation of placental calcium transport. Angiotensin II, acting through the AT1a receptor, is also known to promote placental growth. We examined the effects of bilateral uterine artery and vein ligation (restriction), which mimics placental insufficiency in humans, on growth, intrauterine PTHrP, placental AT1a, and pup calcium. Growth restriction was surgically induced on day 18 of pregnancy in Wistar-Kyoto female rats by uterine vessel ligation. Uteroplacental insufficiency reduced fetal body weight by 15% and litter size (P < 0.001) compared with the control rats with no effect on placental weight or amniotic fluid volume. Uteroplacental insufficiency reduced placental PTHrP content by 46%, with increases in PTHrP (by 2.6-fold), parathyroid hormone (PTH)/PTHrP receptor (by 11.6-fold), and AT1a (by 1.7-fold) relative mRNA in placenta following restriction compared with results in control (P < 0.05). There were no alterations in uterine PTHrP and PTH/PTHrP receptor mRNA expression. Maternal and fetal plasma PTHrP and calcium concentrations were unchanged. Although fetal total body calcium was not altered, placental restriction altered perinatal calcium homeostasis, as evidenced by lower pup total body calcium after birth (P < 0.05). The increased uterine and amniotic fluid PTHrP (P < 0.05) may be an attempt to compensate for the induced impaired placental function. The present study demonstrates that uteroplacental insufficiency alters intrauterine PTHrP, placental AT1a expression, and perinatal calcium in association with a reduction in fetal growth. Uteroplacental insufficiency may provide an important model for exploring the early origins of adult diseases.

parathyroid hormone-related protein; growth restriction; calcium; placenta

INTRAUTERINE GROWTH RESTRICTION affects many human fetuses and contributes significantly to perinatal morbidity and mortality. Epidemiological studies from many populations suggest that a predisposition to adult diseases, including hypertension and diabetes, is associated with low body weight at birth (1). Pregnancies complicated by intrauterine growth restriction are characterized by impaired placental blood flow, increased vascular resistance, and placental dysfunction (22, 39). In Western society, impaired placental blood flow with the associated reduced oxygen and nutrient delivery across the placenta to the fetus is one of the most common features of human pregnancies complicated by growth restriction. Furthermore, in developed countries, many of these babies exhibit asymmetric growth restriction (body weight reduced more than length and "brain sparing"), which can be associated with incomplete postnatal catch-up growth. Experimental placental insufficiency could be more relevant than maternal undernutrition or genetically modified animals as a model to mimic the main type of human intrauterine growth restriction.

The importance of parathyroid hormone-related protein (PTHrP) and its parathyroid hormone (PTH)/PTHrP receptor for normal fetal development has been highlighted by recent gene deletion studies. PTHrP-deficient mice, generated by partial PTHrP gene deletion and homologous recombination, die at birth due to severe skeletal dysplasia, have a reduced maternal-fetal calcium gradient, and have reduced placental calcium transfer (17, 18, 20). Furthermore, mice with homologous deletion of the PTH/PTHrP receptor gene are growth restricted and die midgestation (24). We and others have shown that, during pregnancy, PTHrP may act to stimulate epithelial cell growth and differentiation (25) and placental calcium transport (2, 9, 20), relax uterine smooth muscle (7, 43), and vasodilate fetal-placental vessels (27, 28). Although PTHrP can act in a classical endocrine fashion in humoral hypercalcemia of malignancy, in lactation, and in fetal life, it also exhibits autocrine and paracrine functions through its PTH/PTHrP receptor (34, 48). PTHrP is produced by maternal reproductive and fetal gestational tissues and by many developing fetal tissues (3–6, 10–12, 45, 47). We and others have demonstrated a significant upregulation of PTHrP mRNA in human fetal membranes (3, 11) and PTHrP protein in human (45) and rat (47) amniotic fluid at term compared with at preterm. This is a time when fetal growth and calcium transfer to the fetus are maximal, suggesting important roles for PTHrP in late pregnancy.

Intrauterine growth restriction is thought to result, in part, from reduced transport of oxygen and nutrients across the placenta and reduced fetal-placental blood flow, both of which PTHrP is known to regulate (2, 9, 20, 27, 28). There is also evidence that the placental renin-angiotensin system is chronically activated in intrauterine growth restriction, causing a reduction in AT1 receptor expression within the placenta (26). Angiotensin II increases PTHrP gene expression in heart and aorta (38) and can also stimulate placental PTHrP release (26). We have reported that fetal plasma, placental, and amniotic fluid PTHrP concentrations are reduced in the spontaneously hypertensive rat (SHR), which is considered to be a genetic model of hypertension and growth restriction, compared with its control strain (46, 47). However, in preterm but not in term human growth restriction pregnancies, PTHrP mRNA expression and protein content in fetal membranes are upregulated, presumably as a compensatory mechanism (5). Although many perinatal factors are suggested to be involved in the development of intrauterine growth restriction, calcium is known to play an important role. PTHrP is involved in the regulation of calcium in the microenvironment and has specific effects on cellular growth (19, 29, 34). Placental calcium transfer is reduced in the growth-restricted rat fetus induced by bilateral uterine artery and vein ligation (30) and in the PTHrP gene knockout mouse (20). Thirty to forty percent of low birth weight infants, including those from human pregnancies complicated by growth restriction, have neonatal hypocalcemia (40), which may be a consequence of impaired placental calcium transport.

We have used the accepted model of perinatal growth restriction in rats caused by bilateral uterine artery and vein ligation first described by Wigglesworth (42) that mimics late-gestation placental insufficiency in humans. This model is also characterized by asymmetric growth restriction and incomplete postnatal catch-up growth. The primary aim of this study was to investigate the effects of uteroplacental insufficiency induced by uterine vessel ligation on fetal growth and intrauterine PTHrP and calcium by quantifying fetal and maternal plasma and amniotic fluid PTHrP and calcium concentrations. Furthermore, placental and uterine PTHrP tissue content and PTHrP and PTH/PTHrP receptor and placental AT1a mRNA expression were also examined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental protocol. Wistar-Kyoto rats (9–13 wk of age) were obtained from the Australian Resource Centre (Murdoch, WA, Australia) and fed standard food pellets and tap water ad libitum. Rats were housed in a 12:12-h light-dark cycle (lighting from 0600 to 1800) and a temperature-controlled room at 19–22°C. Rats were mated after the vaginal impedance reader (model MK-10B, MuKomachi Kikai, Osaka, Japan), which measures the electrical impedance of the epithelial cell layer of the vaginal mucosa, in the afternoon indicated that they were in proestrus and presumably in estrous that night. The presence of sperm in the vaginal smear the following morning was taken as day 1 of pregnancy. This study was approved by The University of Melbourne Animal Experimentation Ethics Subcommittee.

On day 18 of gestation, pregnant rats were randomly allocated into the restricted group (n = 19) or sham control group (n = 20) group. Uterine vessel ligation was carried out to induce uteroplacental insufficiency and intrauterine growth restriction (42). Rats were anesthetized by tail vein intravenous injection of a mixed solution containing ketamine (Parnell Laboratories, Alexandria, NSW, Australia; 50 mg/kg of body wt) and Ilium Xylazil-20 (Troy Laboratories, Smithfield, NSW, Australia; 10 mg/kg of body wt). Surgery in the restricted group involved a midline abdominal incision and exposure of only the cervical end of the uterus with clear access to the uterine vessels. After ligation of the uterine vessels on both left and right sides using 4-0 silk suture, the surface of the uterus was flushed with sterile saline and placed back into the body cavity. Sham surgery for the control group was performed in the same manner except the uterine vessels were not ligated. All surgical procedures were performed under aseptic conditions. The duration of surgery was 10 min, and length of anesthesia was ~40 min.

Tissue and fluid collection. On the morning of day 20 of pregnancy, pregnant rats were anesthetized intraperitoneally with pentobarbital sodium (Nembutal, Boehringer Ingelheim, Sydney, Australia; 120 mg/kg body wt). The uterus was exteriorized and weighed, and embryonic sacs were separated from the uterus and also weighed. Maternal blood was obtained by cardiac puncture. The numbers of implantation sites, live viable pups, and dead pups were counted. Amniotic fluid was aspirated with a 25-gauge needle. Fetal blood was obtained after decapitation with ammonium heparinized capillary tubes and was pooled within a litter. Blood was placed in heparinized Eppendorf tubes for PTHrP and calcium analyses. Aprotinin (5.2 TIU/mg solid; Sigma, St. Louis, MO) was added to the tubes used for PTHrP analysis. Amniotic fluid from every third fetus was collected into aprotinin (5.2 TIU/mg solid; Sigma); all other amniotic fluid samples were untreated. Placentas and fetal membranes were separated and weighed individually. Fetal body weight and crown rump length, head width, and hindlimb length were measured with calipers. Ponderal index was calculated as body weight (in g) x 100/crown rump length (in cm3), as another indicator of growth incorporating weight and length. One fetus per litter was snap frozen for whole body calcium analysis and stored at –20°C. Amniotic fluid volume was derived by subtracting placental, fetal membrane, and fetal weights from total amniotic sac weight. Placenta, fetal membrane, and uterine tissue samples were frozen in liquid nitrogen and stored at –80°C until analyzed. One placenta per litter was immersion fixed horizontally in 10% neutral-buffered formalin and was subsequently embedded in paraffin for immunohistochemistry. Maternal and fetal blood was centrifuged at 2,400 rpm at 4°C for 15 min, and the plasma fraction was removed. Amniotic fluid and plasma samples were frozen in liquid nitrogen and stored at –20°C until analyzed.

Protein extraction and protein assay. Samples of frozen placental (n = 14 for control group, n = 12 for restricted group) and uterine (n = 6 for control group, n = 4 for restricted group) tissue (1.0 g) were homogenized for 20 s at 24,000 rpm in 5 ml of acetic acid (1 M) using previously established techniques (47). Duplicate 500-µl aliquots of the homogenate were removed for protein assay. The remaining homogenate was incubated for 2–3 h at 4°C and centrifuged at 20,000 g for 15 min at 4°C. The supernatant was then dialyzed against 5 liters of deionized water for 22–26 h at 4°C using Spectra-Por 3 dialysis tubing (molecular weight of 6,000–8,000; Cole Palmer, Niles, IL) as previously described (47). The extract was stored at –20°C for PTHrP radioimmunoassay.

Placental morphological development. The placenta paraffin-embedded blocks were sectioned at 10 µm and stained with Masson’s trichrome. At least two sections a minimum of 50 µm apart were analyzed per restricted (n = 5) and control (n = 3) placentas. Sections were examined with a 1x objective lens and a 10x ocular lens on an Olympus BH2 microscope equipped with a video image analysis system using Image Pro software (version 4.5, Media Cybernetics). The proportion of placental area that was labyrinthine (exchange region) and diameter (or largest width) of placentas at their widest point were calculated.

PTHrP and calcium measurements. Plasma, amniotic fluid, and tissue concentrations of PTHrP were quantified by a sensitive NH2-terminal radioimmunoassay that does not recognize PTH (14, 47). The radioimmunoassay uses a polyclonal goat antiserum against synthetic PTHrP(1–40) and recombinant PTHrP(1–84) as standard. The detection limit was 2 pmol/l, and intra- and interassay coefficients of variation were 4.8 and 13.6%, respectively. We determined total calcium concentrations on frozen samples using a Beckman Synchron CX-5 clinical system (Beckman Coulter, Fullerton, CA). We measured ionic calcium concentrations using ion-selective electrodes correcting for pH (Radiometer ABL615 blood-gas/electrolyte analyzer, Copenhagen, Denmark) from fresh amniotic fluid and fetal and maternal plasma samples. Total body calcium was also measured in a separate group of pups killed on day 6 postnatal (n = 8 in control group, n = 6 in restricted group) after placental restriction on day 18 of pregnancy, whose pup body weights were significantly lower in restricted animals (1.5 ± 0.02 g) compared with control animals (1.7 ± 0.02 g). Stored frozen pups were placed in porcelain crucibles and exposed to 600°C (Tetlow Furnace, Melbourne, Victoria, Australia) for 12 h. This ash solution was analyzed for total calcium content using the Beckman CX-5 analyzer (41).

Gene expression analysis: isolation of total RNA and DNase treatment of total RNA. We extracted total RNA from frozen placental (n = 8 in control group, n = 9 in restricted group) and uterine (n = 9 in control group, n = 6 in restricted group) tissues using QIAGEN RNeasy kits (Qiagen, Clifton Hill, Victoria, Australia). For each total RNA sample, 10 µg were DNase treated with "DNA-Free" (Ambion, Austin, TX). The reactions were incubated at 37°C for 20 min before an inactivation step at room temperature for 2 min followed by centrifugation at 10,000 g for 1 min to pellet the inactivation reagent, leaving the DNA-free RNA. The total RNA quality and content were established after we obtained absorbance readings at 260 and 280 nm. Samples were stored at –80°C until further use.

cDNA synthesis. Each cDNA synthesis reaction consisted of 10x RT buffer, 5.5 mM MgCl2, 500 µM of each dNTP, 50 ng of random hexamers, 1 µl of RNase-OUT ribonuclease inhibitor, 50 U of SuperScript II, and 1 µg of total RNA. All of the above reagents were supplied in a SuperScript first-strand synthesis kit (Invitrogen Life Technologies, Carlsbad, CA). The reverse transcription reactions were performed in a cooled/gradient palm cycler (Corbett Research, Mortlake, NSW, Australia) with incubations at 25°C for 10 min, 42°C for 50 min, and 70°C for 15 min. Reactions were collected by brief centrifugation before addition of 1 µl RNase H and incubation for 20 min at 37°C. Samples were stored at –80°C. To ensure there was no contaminating genomic DNA, a separate reverse transcription reaction was performed for each sample that did not contain the SuperScript II.

Real-time PCR. We performed real-time PCR (15) using a Rotor-gene 3000 (Corbett Research). Primers and TaqMan probes for real-time PCR were designed with the use of Primer Express version 1.5 (Applied Biosystems). The nucleotide sequences for the PTHrP or PTH/PTHrP receptor primers and probes are listed in Table 1; their positions relative to GenBank/EMBL data entries are also provided in Table 1. Ribosomal 18S (18S) TaqMan probe and primers were supplied by Applied Biosystems. The intra-assay coefficients of variation for these genes are 2.5% (PTHrP) and 2.1% (PTH/PTHrP receptor).


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  		Table 1. Real-time PCR primer and probe

 
For the relative quantitation of gene expression, a comparative threshold cycle (CT) method was employed. A CT value reflects the cycle number at which fluorescence is first detected. For each TaqMan probe, 6-carboxy-fluorescein was attached to the 5' end for PTHrP, PTH/PTHrP receptor, or AT1a as well as for the 18S. For 18S, AT1a, and PTH/PTHrP receptor probes, 6-carboxytetramethylrhodamine was attached to the 3' end. The PTHrP probe was an MGB probe. PTHrP or PTH/PTHrP receptors were run in separate tubes to the endogenous control. The AT1a gene was run in a multiplex reaction with 18S. A validation experiment was performed to ensure that a similar CT value was obtained when the AT1a and 18S were in separate reactions compared with the multiplex reaction. A validation experiment was also performed to test whether the comparative CT method could be used for the relative quantitation of gene expression. Equal PCR efficiencies of PTHrP, PTH/PTHrP receptor, or AT1a amplifications and 18S were obtained when different template concentrations (0.05–50 ng) were assayed in separate tubes.

PCR reactions were carried out in 25-µl volumes consisting of 1x Platinum qPCR Sper-Mix-UDG (Invitrogen Life Technologies), 250 nM 18S TaqMan probe, 100 nM 18S forward primer, 300 nM reverse primer, and PTHrP, PTH/PTHrP receptor, or AT1a and TaqMan probe, where the final concentrations are provided in Table 1. We amplified cDNA (40 ng) and no reverse transcriptase preparations using the following conditions: 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. All placental and uterine samples from both groups were analyzed on the same run, and duplicate runs were performed.

Real-time PCR calculations. Comparative CT calculations for the expression of PTHrP, PTH/PTHrP receptor, or AT1a were all relative to a chosen calibrator (control placenta). To achieve quantitative values, 18S CT values were first subtracted from the CT value for each well to give a {Delta}CT value. {Delta}{Delta}CT values were achieved by subtracting the average calibrator {Delta}CT value from the {Delta}CT value. The expression of PTHrP, PTH/PTHrP receptor, or AT1a relative to the calibrator was evaluated using the expression 2–{Delta}{Delta}CT.

Statistical analyses. Homogeneity of variance was analyzed with the use of Bartlett’s test. Data were analyzed by t-test. Intrauterine weights and dimensions were averaged within a litter and analyzed. Data are presented as means ± SE, and P < 0.05 was taken as statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Body and organ weights. Maternal body (278 ± 6 g for control, 261 ± 5 g for restricted) and uterine weights were not different between the groups. Figure 1 illustrates fetal body weight and litter size of viable fetuses as measured at day 20 of gestation. A 15% reduction in fetal body weight and 21% reduction in litter size of viable fetuses were observed in the restricted rats compared with control rats (P < 0.001; Fig. 1) despite a similar number of implantation sites (containing live or dead pups). The effect on survival was a generalized one because percent survival rates were similar between the ovarian and cervical portions of the uterus. Placental weight was not different between the groups, but fetal-to-placental weight ratio was significantly lower in the restricted group (P < 0.0001; Table 2). Uteroplacental restriction was associated with a trend to an increase in placental total surface area (P = 0.08; Table 2) and an increase in diameter (P < 0.05; Table 2) but did not alter the proportion of placental area or diameter associated with the labyrinthine zone. Crown rump length, but not ponderal index, head width, or hindlimb length, was lower in the restricted compared with control fetuses (P < 0.05; Table 2). Total amniotic sac weight (fetus, placenta, amniotic fluid, and fetal membranes) and fetal membrane weight were significantly lower in restricted compared with control animals (P < 0.01; Table 2). Amniotic fluid volume was not different between the groups (Table 2). Fetal weight (1.97 ± 0.04 g) and placental weight (0.40 ± 0.008 g) of unoperated control animals [previously published by our laboratory (47)] were significantly higher than sham-operated controls (P < 0.05), reflecting a developmental delay caused by surgical stress consistent with a previous report (31).



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  		Fig. 1. Fetal weight (averaged within a litter before analysis) on day 20 of gestation in the control (open bar; n = 20) and restricted (closed bar; n = 19) (means ± SE) rats. Fetal weight was significantly lower in the restricted group compared with control (*P < 0.002).

 

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  		Table 2. Weights and dimensions of control and restricted rats

 
PTHrP and calcium measurements. There were no significant differences in maternal plasma PTHrP, ionic calcium, and total calcium concentrations between the two groups (Table 3). Fetal plasma PTHrP and ionic calcium concentrations were not different between control and restricted animals (Table 3). Fetal plasma ionic calcium levels were higher than those in maternal plasma (Table 3). Fetal whole body total calcium content was not different between the groups on day 20 of gestation (Table 3). In a separate study, pup whole body total calcium content on day 6 after birth was significantly lower after placental restriction compared with control (P < 0.01; Table 3). Amniotic fluid PTHrP concentrations (P < 0.05; see Fig. 3) and content (7.9 ± 0.4 fmol for control, 9.2 ± 0.4 fmol for restricted; P < 0.05) were significantly higher in the restricted group compared with the control group. Amniotic fluid ionic calcium and total calcium concentrations were not different between the groups (Table 3).


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  		Table 3. PTHrP and calcium measurements

 


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  		Fig. 3. Amniotic fluid PTHrP concentrations (A; n = 14 control; n = 12 restricted) and uterine PTHrP content (B; n = 6 control; n = 4 restricted) on day 20 of gestation in control (open bars) and restricted (closed bars) (means ± SE) rats. Amniotic fluid PTHrP concentrations and uterine PTHrP content were significantly higher in the restricted group compared with the control group (*P < 0.05).

 
Placental and uterine tissue content and mRNA expression. Placental PTHrP tissue content was significantly reduced by 46% in the restricted group compared with the control group (P < 0.05; Fig. 2). There were significant increases in PTHrP (by 2.6-fold), PTH/PTHrP receptor (by 1.6-fold), and AT1a (by 1.7-fold) relative mRNA in placenta in the restricted group compared with the control group (P < 0.05; Fig. 2).



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  		Fig. 2. Placental AT1a (A), parathyroid hormone-parathyroid hormone-related protein (PTH/PTHrP) receptor (B), and PTHrP (C) relative mRNA gene expression (n = 8 control; n = 9 restricted) and placental PTHrP content (D; n = 14 control; n = 12 restricted) on day 20 of gestation in control (open bars) and restricted (closed bars) (means ± SE) rats. Placental PTHrP content was significantly lower, whereas gene expression was significantly higher in the restricted group compared with the control group (*P < 0.05).

 
Restricted uterine PTHrP tissue content was significantly elevated by 2.5-fold above control (P < 0.05; Fig. 3). There were no differences in PTHrP (1.30 ± 0.30 for control group, 1.09 ± 0.20 for restricted) relative mRNA expression, AT1a (1.08 ± 0.12 for control, 1.12 ± 0.27 for restricted) relative mRNA expression, and PTH/PTHrP receptor (1.26 ± 0.31 for control, 1.36 ± 0.47 for restricted) relative mRNA expression in uterus between control (n = 9) and restricted (n = 6) groups.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The degree of fetal growth restriction (15%) and reduced litter size of viable fetuses are consistent with previous studies that have shown that uteroplacental insufficiency reduces oxygen and nutrient delivery to the fetus causing growth restriction (8, 23, 30, 31, 33, 35–37). The number of implantation sites was not different between the groups, and the survival rates were similar at the ovarian and cervical ends of the uterus, a finding that is consistent with a generalized effect of reduced oxygen and nutrient supply in utero on both fetal survival and growth. Fetal weight and crown rump length were reduced without large changes in head width (as an indicator of brain growth with a brain-sparing effect), which is indicative of asymmetric growth restriction previously reported in this model (8, 32). The maintenance of placental weight [with a small increase in placental area (P = 0.08) and an increase in placental length] in association with fetal growth restriction is in agreement with one (37) but not another (30) previous study. This finding suggests the occurrence of preserved placental growth in the face of reduced perfusion and function. Interestingly, amniotic fluid volume, which has not been investigated previously in this model, was not altered at this stage of pregnancy in response to placental insufficiency. This supports the notion that fetal urine production, which is a major contributor to amniotic fluid volume, and amniotic fluid volume regulation were not influenced 2 days after the insult.

Placental insufficiency, induced by bilateral uterine vessel ligation, reduced oxygen and nutrient delivery to the fetus and lowered placental PTHrP content by 46%. This large decrease in placental PTHrP content was associated with a substantial increase in PTHrP or PTH/PTHrP receptor mRNA expression (by 2.6- and 11.6-fold, respectively) following uteroplacental restriction. Although real-time PCR is considered to be more sensitive and accurate than protein measurements, the large changes in gene expression and protein content are unlikely to be associated with methodological problems. The lower protein content would be expected to upregulate receptor expression in an attempt to overcome the deficit. The reason for the high PTHrP gene expression in the face of lowered protein is unclear, and measurements on additional days after placental restriction may provide further insight. That both PTHrP and AT1a mRNA expression were upregulated may suggest that, in response to reduced placental perfusion, factors that promote placental growth increase in an attempt to preserve placental function as indicated by the increase in placental diameter. Whether the increased placental AT1a mRNA expression, through its vasoconstrictive actions, further compromises placental perfusion remains to be established. The increased AT1a expression could also reflect a decrease in angiotensin II production by the placenta, which in turn may have contributed to the decrease in PTHrP content as angiotensin II is known to stimulate placental PTHrP production (26).

The decrease in placental PTHrP content could, together with the effects of uteroplacental insufficiency, contribute directly to the compromised fetal growth by its known effects on placental vasodilatation (27, 28) and calcium transport (2, 9, 20). We suggest that the impaired placental function and lower PTHrP levels may have contributed to an impaired placental calcium transport, although 2 days of uteroplacental insufficiency may not have been sufficient to detect changes in fetal plasma and subsequently in fetal bone and total body calcium. With the use of the same rat uteroplacental insufficiency model, previous work has shown a reduction in total fetal body calcium content (30) associated with reduced maternofetal 45calcium transfer; however, this was not detected until 3 days after induction (30). Supporting our suggestion that placental insufficiency, possibly by PTHrP, alters calcium homeostasis is that pup total body calcium content 6 days after birth was significantly reduced in the restricted group compared with the control group. The relative roles of lowered placental calcium transport late in gestation and impaired mammary function with lower milk calcium in early postnatal life as well as possible alterations to pup calcium homeostasis in mediating the lower postnatal total body calcium remain to be determined. Knockout studies (20, 21) have indicated that PTHrP-directed calcium transport and fetal PTH are the major regulators of circulating fetal calcium levels. We did not have the opportunity to measure fetal PTH concentrations (due to insufficient blood volume for analyses). The lack of effect on maternal and fetal plasma PTHrP and calcium at this stage of gestation suggests that the uteroplacental restriction directly influenced placental PTHrP and that there may be independent regulation of PTHrP in the fetus at the time of sampling. Previous studies have reported lower fetal plasma, amniotic fluid, and placental PTHrP concentrations in the growth-restricted SHR, which is a genetic, not a placental restriction, model of growth restriction (47). It remains to be determined whether these reductions were due to the genetic makeup of the SHR or the growth restriction itself.

Uteroplacental insufficiency resulted in altered uterine PTHrP tissue content, which was increased by 2.5-fold, in the absence of changes in PTHrP or PTH/PTHrP gene expression. The reason for this is not clear, but it may reflect changes in protein turnover or a transient increase in gene expression altering PTHrP tissue content. It is also possible that uteroplacental insufficiency induced cell death, which could account for the increased PTHrP (16). Alternatively, a compensatory mechanism could have come into play to promote uterine blood flow to protect the fetus from the placental insufficiency insult. This is consistent with reports of transient reductions of uterine blood flow (13) and fuel availability (31) following uteroplacental insufficiency in the rat. Future studies at different time points after uteroplacental restriction could provide further information regarding the ontogeny of gene and protein alterations in the placenta and uterus. It has been suggested that uterine PTHrP (which was significantly raised after restriction) as well as placental, fetal membrane, and fetal plasma may contribute to amniotic fluid PTHrP (47), thus providing an explanation for the raised amniotic fluid levels. It is not yet certain whether amniotic fluid PTHrP contributes to fetal growth through its actions on cellular growth and differentiation (25) or whether it contributes to placental calcium transfer and blood flow. Thus we can only speculate about the consequences of the raised amniotic fluid PTHrP levels. Our recent study (44) explored the potential for PTHrP to act as a growth promoter in the SHR genetic model of growth restriction (44). When endogenous PTHrP is increased in uterus, placenta, and fetal circulation, following PTH/PTHrP receptor antagonist infusion, the SHR fetal weight is increased significantly (44). Effects on fetal weight were not observed when uterine PTHrP was elevated in the absence of placental and fetal plasma changes, indicating that a generalized substantial increase in intrauterine PTHrP is required to enhance fetal growth (44). We have previously reported (5) that preterm, but not term, human intrauterine growth restriction is associated with an upregulation of placental and fetal membrane PTHrP. Studies at term and at other stages of pregnancy after rat uteroplacental insufficiency may provide insight into the etiology of intrauterine PTHrP changes in relation to the human data.

Our placental insufficiency phenotype of reduced fetal weight, in association with lower placental PTHrP and lower perinatal calcium, is consistent with that described for the PTHrP knockout mouse (20, 21), except that a complete lack of PTHrP is postnatally lethal. An upregulation of gene expression of factors (PTHrP) that promote placental growth and their receptors (PTH/PTHrP and AT1a) may contribute to further detrimental effects of reduced placental perfusion. Understanding the consequences of placental insufficiency and its impact on growth and calcium on later growth and development may provide an important animal model to study the early origins of adult cardiovascular disease and diabetes.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by a National Health and Medical Research Council Project Grant (to M. E. Wlodek) and a National Health and Medical Research Council Program Grant (J. M. Moseley).


   ACKNOWLEDGMENTS
 
We acknowledge the support for animal management provided by Damaris Delgado and the Biological Research Facility staff. We thank Prof. Geoffrey Tregear and Angela Gibson (Howard Florey Institute of Experimental Physiology and Medicine) for measuring total calcium concentrations and thank Patricia Ho (St. Vincent Institute of Medical Research) for performing the PTHrP radioimmunoassays.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. E. Wlodek, Dept. of Physiology, Univ. of Melbourne, Victoria 3010, Australia (E-mail: m.wlodek{at}unimelb.edu.au)

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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