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BOR - Papers in Press, published online ahead of print December 26, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.023853
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BIOLOGY OF REPRODUCTION 70, 1277–1285 (2004)
DOI: 10.1095/biolreprod.103.023853
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Maternal Growth Hormone Treatment from Day 35 to 80 of Gestation Alters Nutrient Partitioning in Favor of Uteroplacental Growth in the Overnourished Adolescent Sheep1

Jacqueline M. Wallace2, John S. Milne, and Raymond P. Aitken

Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Overnourishing the pregnant adolescent ewe promotes maternal tissue synthesis at the expense of placental growth and leads to a major reduction in lamb birth weight at term. Growth hormone (GH) secretion is attenuated in these overnourished dams and the maternal somatotrophic axis may play a key role in coordinating nutrient usage in the pregnant adolescent. Thus we investigated whether increasing maternal GH during the period of rapid placental proliferation alters nutrient partitioning between the maternal, placental, and fetal tissues as assessed at Day 81 of gestation. Adolescent recipient ewes were implanted with singleton embryos, derived from superovulated dams and a single sire on Day 4 postestrus. Thereafter, the ewes were offered either a high (H) or moderate intake (M) of the same complete diet. From Day 35 to 80 of gestation, ewes were either injected twice daily (s.c. at 0800 and 1800 h) with recombinant bovine GH (bGH, 0.14 mg/kg live weight/day) or remained untreated (n = 8 ewes per group). Maternal concentrations of GH, insulin, insulin-like growth factor (IGF-1), glucose, and non-esterified fatty acids (NEFAs) were higher, and leptin secretion lower, in bGH-treated dams from both nutritional groups. Maternal body weight gain was higher in H versus M groups and was independent of bGH treatment. Treatment with bGH reduced relative perirenal and carcass fat deposition and increased carcass protein content in both H and M dams. Uteroplacental mass (uterus + placentomes + fetal membranes) averaged 1099, 1069, 1112, and 1754 g in M, H, M+GH, and H+GH groups. This significant increase in uteroplacental development in the H+GH group was associated with higher fetal kidney and liver weights and elevated fetal insulin, glucose, and lactate concentrations. Treatment with bGH also induced polyhydramnios in the H group. The transplacental glucose gradient was increased twofold in the H+GH group but placental GLUT- 1 and GLUT-3 expression was unaffected. In conclusion, administration of GH during the period of rapid placental proliferation alters endocrine status and thus nutrient partitioning in the overnourished adolescent dam in favor of uteroplacental and fetal growth. It remains to be established whether these effects are due wholly to alterations in maternal metabolism or if they also reflect an effect of bGH and/or the IGF system at the level of the uteroplacenta.

female reproductive tract, growth hormone, pregnancy, placenta, placental transport


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pregnancy represents one of the most anabolic periods of the female life cycle, and appropriate nutrient partitioning between the maternal body and gravid uterus throughout gestation is essential to ensure optimum fetal growth and neonatal survival. When pregnancy coincides with the growth of the mother, the normal hierarchy of nutrient partitioning may be altered at the expense of nutrient delivery to the fetus. Data from the Camden Adolescent Pregnancy and Nutrition Project demonstrates that up to 50% of human adolescents continue to grow while pregnant and, in spite of larger pregnancy weight gains and increased fat stores, the average birth weight of their babies is reduced compared with nongrowing adolescents' babies [1]. We initially developed a highly controlled animal paradigm to examine the role of maternal nutrition and growth status on the etiology of poor pregnancy outcome during adolescence. Using this paradigm, we have consistently demonstrated that overnourishing the singleton-bearing adolescent sheep promotes rapid maternal growth throughout pregnancy at the expense of the nutrient requirements of the gravid uterus. This results in a major reduction in placental mass and a significant decrease in lamb birth weight at term relative to slow-growing, M-intake adolescent dams of equivalent gynecological age [2, 3]. Rapid maternal growth is also associated with higher spontaneous abortion rates in late gestation and, for ewes delivering live young, with a reduction in gestation length [3]. Thus, our paradigm replicates the key features of human adolescent pregnancy, namely an increased risk of abortion, preterm delivery, and low birth weight [4–6].

The maternal somatotrophic axis may play a role in coordinating nutrient use between maternal, placental, and fetal tissues in the growing adolescent animal. We have previously demonstrated that, in overnourished adolescent sheep, maternal insulin and insulin-like growth factor (IGF- 1) concentrations are elevated from early in gestation and it is probable that these hormones provide a sustained anabolic stimulus to maternal tissue deposition, primarily of adipose tissue [7, 8]. In contrast, growth hormone (GH) pulse frequency and mean concentrations during mid and late gestation are lower in overnourished compared with moderate intake dams and inversely related to maternal insulin concentrations [7]. In humans, intrauterine growth restriction is associated with lower than normal concentrations of GH in the maternal circulation and reduced placental GH mRNA expression in the term placenta [9, 10]. Similarly, GH deficiency in rat dams is associated with impaired fetal growth [11]. GH does not cross the placenta, but increased maternal GH concentrations may influence fetal growth by inhibiting the lipogenic action of insulin and increasing glucose concentrations in the maternal circulation and/or by increasing placental capacity to transfer nutrients to the fetus [12]. Alternatively, GH of pituitary or extrapituitary origin may influence the growth of the uteroplacenta directly or indirectly via the IGF system. Ovine placental GH has been detected in the placentome between Days 30 and 75 of pregnancy, while GH receptor mRNA is expressed in the endometrium and placentome throughout pregnancy [13]. Furthermore, the various components of the IGF system have been localized in the ovine uteroplacenta, where they express spatial and temporal patterns of expression [14], some of which are sensitive to maternal nutritional status [15]. In spite of these observations, previous studies of the effect of maternal GH administration on placental and/or fetal growth in normal pregnancies are equivocal. Treatment of pregnant ewes with recombinant bovine GH (bGH) for 14 days from Day 70 to 83 or Day 98 to 111 of gestation did not influence placental size, but the later period of treatment was associated with increased fetal weight, as determined at Day 112 of gestation [16]. In contrast, treatment between Days 97 and 124 of pregnancy did not influence fetal weight at Day 125 or at term [17]. These treatments were unlikely, however, to have a direct impact on placental growth, as the maximal rate of placental proliferation in the sheep occurs between Days 50 and 60 of gestation, with an abrupt cessation of DNA and mass accumulation between Days 75 and 80 of gestation [18]. Studies in the polytocous pig have demonstrated that, when treatments are applied during early pregnancy, both placental and fetal weights are increased [19], particularly in the lowest fetal weight quartile [20].

Recent studies in late gestation clearly demonstrate that it is the small size of the placenta per se rather than alterations in placental nutrient uptake, metabolism, and transport that limit fetal growth in the overnourished adolescent [21, 22]. The aim of the present study was, therefore, to determine whether treatment with recombinant bGH during the period of rapid placental proliferation could alter nutrient partitioning between the maternal, placental, and fetal compartments of rapidly growing adolescent sheep.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Experimental Design

All procedures were licensed under the UK Animals (Scientific Procedures) Act of 1986 and by the Rowett Research Institute's Ethical Review Committee.

Embryos from superovulated adult ewes (Border Leicester x Scottish Blackface), inseminated by a single sire, were recovered on Day 4 after estrus and transferred synchronously singly into the uterus of recipient ewe lambs (Dorset Horn x Mule) exactly as described previously by Wallace et al. [7]. Embryo transfer was carried out during the midbreeding season and the animals were housed in individual pens under natural lighting conditions at the Rowett Research Institute (57°N, 2°W). At the time of embryo transfer, the recipient ewe lambs were peripubertal (~7 mo old), with a mean live weight of 46 ± 0.6 kg and a body condition score of 2.3 ± 0.02 U. Immediately following embryo transfer, recipient ewe lambs were evenly allocated to one of two nutritional treatments on the basis of live weight, body condition score, and ovulation rate at the time of transfer. Where possible, care was also taken to randomize the maternity of the embryo. Recipients were individually offered either a high (n = 24) or moderate (n = 24) level of a diet calculated to promote rapid or low maternal growth rates, respectively. The moderate dietary level was in fact a control group in that this level of dietary intake was predicted to optimize placental and fetal growth in this genotype. The complete diet supplied 10.2 MJ metabolizable energy and 137 g crude protein per kg and was offered in two equal feeds at 0800 and 1600 h daily. The diet contained 30% (w/w) coarsely milled hay, 50% barley, 10% molasses, 9% fishmeal, 0.3% salt, 0.5% dicalcium phosphate, and 0.2% of a vitamin- mineral supplement and had an average dry matter content of 86%. Animals offered moderate intakes received their entire ration immediately, while those offered high intakes had the level of feed gradually increased over a 2–3-wk period until the level of daily feed refusal was approximately 15% of the total offered (equivalent to ad libitum intakes). The level of feed offered was reviewed three times weekly and adjusted, on an individual basis as and when appropriate, on the basis of body weight- change data (recorded weekly) and the level of feed refused (recorded daily). Body condition score was subjectively assessed on a five-point scale (1 = emaciated, 5 = obese; [23]).

Pregnancy rate was initially estimated by measurement of plasma progesterone concentrations at Day 18 after estrus (Day 14 after embryo transfer) and half the recipients that conceived in each nutritional treatment group were further allocated evenly (on the basis of liveweight gain from embryo transfer to Day 34) to receive GH. This yielded four groups: high (H), high + GH (H+GH), moderate (M), moderate + GH (M+GH). Recombinant bGH (Sometribove; gifted by Monsanto Company, St. Louis, MO) was administered twice daily (0800 and 1800 h) from Day 35 to 80 of gestation at a dose rate of 0.14 mg/kg/day. The bGH was solubilized in 0.035 mM sodium bicarbonate buffer (pH 9.5) and administered s.c., with care taken to alternate the injection sites on either side of the neck. Individual dose volumes (<2 ml) were adjusted weekly on the basis of current live weight throughout the treatment period. The untreated groups were temporarily restrained twice daily and all ewes rapidly adapted to being handled. Transabdominal ultrasound at approximately Day 45 of gestation confirmed viable fetuses in 8, 9, 8, and 8 ewes in the H, H+GH, M, and M+GH groups, respectively. A further ultrasound examination on approximately Day 75 of gestation checked fetal viability before autopsy.

Measurements at Autopsy

On Day 81 of gestation, ewes received their morning feed, were weighed and scored for body condition score, and were then killed by i.v. injection of an overdose of sodium pentobarbitone (20 ml Euthesate, 200 mg pentobarbitone/ml; Willows Francis Veterinary, Crawley, UK) and exsanguination (by severing the main vessels of the neck). The gravid uterus was removed, weighed, and opened, and a fetal blood sample was collected by cardiac puncture immediately before intracardiac administration of sodium pentobarbitone (3 ml Euthesate) to kill the fetus. After the cord was clamped, the fetus was removed, towel dried, and weighed. The biparietal head diameter, crown-rump length, and girth at the umbilicus were measured and the major fetal organs dissected and weighed. Twelve intact placentomes (representative of both size and type of the remaining placentomes and from both uterine horns) were removed for analyses (not reported in this article). For the remaining placental tissues, the intact placentomes were dissected and their number and weight recorded (represents ~85% of the tissue originally available). These placentomes were then dried at 100°C for 72 h and the dry weights recorded. The chorioallantoic membranes and residual uterus were weighed and the volume of the combined amniotic and allantoic fluids recorded.

The maternal liver and perirenal fat was dissected and weighed. The maternal body was divided into carcass and noncarcass components and the chemical composition of the carcass was determined after removing the pelt, head, and feet. The carcasses were individually homogenized in a Wolfking (Slagelse, Denmark) mincer [24]. Three samples of each homogenate were freeze dried, ground in a laboratory mincer, and subsampled for analyses. The residual moisture content of the carcass remaining after freeze drying was obtained by drying to constant weight at 100°C. The fat content was determined by the chloroform-methanol technique [25] and the nitrogen by the Kjeldahl method [26]. The ash content of the samples was determined by ashing at 600°C to constant weight.

Blood Sampling and Biochemical Analysis

Blood samples were collected, between 1100 and 1200 h, from all ewes by jugular venepuncture weekly during Weeks 1–4 of pregnancy and twice weekly thereafter. These samples were analyzed for GH, insulin, and glucose. A further maternal blood sample collected on the morning of autopsy was additionally analyzed for IGF-1, leptin, non-esterified fatty acid (NEFA), and alpha-amino N. Fetal blood samples were analyzed for insulin, IGF-1, glucose, and lactate. GH, insulin, and IGF-1 concentrations were measured in duplicate by radioimmunoassays described previously [7, 27, 28]. The limit of detection was 1 ng GH/ml, 4 µU insulin/ml, and 6 pmol IGF-1/ml. The antiserum used in the ovine GH radioimmunoassay cross-reacts with both bGH (99.5%) and recombinant bGH (17.3%). Leptin concentrations were determined as previously described using an ovine- specific solid-phase sandwich ELISA [29], the sensitivity of which was 1 ng leptin/ml. The intra- and interassay coefficients of variation were 3.1% and 3.7% for GH and 3.5% and 7.5% for insulin. The intraassay coefficient of variation for IGF-1 was 13.5% and for leptin was 12.5%. Plasma glucose and lactate concentrations were measured in duplicate with a Yellow Springs Instruments (YSI; Yellow Springs, OH, USA) dual biochemistry analyzer (model 2700). The YSI instrument was calibrated with known standards after every fourth determination. Plasma NEFA and alpha-amino N concentrations were determined as originally described by Matsubara et al. [30] and Palmer and Peters [31].

Quantitative Real-time Reverse Transcription-Polymerase Chain Reaction

Messenger RNA levels for the glucose transporters GLUT-1 and GLUT-3 were determined using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from an individual representative whole placentome from each pregnancy using Tri Reagent (Sigma Chemical Co., St. Louis, MO). The quality and quantity of total RNA was determined via capillary electrophoresis using an Agilent 2100 Bioanalyzer (Agilent Technologies, Wilmington, DE). All reagents and procedures used for the real-time RT-PCR were purchased and used as described by Applied Biosystems [32]. For each sample, approximately 30 ng total RNA was reverse transcribed in triplicate using TaqMan Reverse Transcription Reagents and MultiScribe Reverse Transcriptase. Taqman probes and primers sets were designed from species- specific sequences of genes using Primer Express Software. For GLUT-1, the sequences of the cDNA forward and reverse primers were 5'- TGCTCATTAACCGCAACGAG-3' (335–354, GenBank U89029) and 5'- GGTCCCACGCAGCTTCTTC-3' (378–396), respectively. The sequence of the FAM (5-carboxyfluorescein)-labeled Taqman probe was 5'-AGAACCGGGCCAAGAGCGTGC-3' (356–376). For GLUT-3, the sequence of the forward and reverse primers were 5'-TTTGGAAGAACGGTCAGAAACA-3' (250–271, GenBank L39214) and 5'-CAGACAAGGACCACAGGGATG-3' (293–313), respectively, while that of the FAM-labeled probe was 5'-CCCCGTCCAGCGTGCTCCTC-3' (272–291). Polymerization and amplification reactions for each RT were carried out in duplicate in 96-well PCR plates in a final volume of 10 µl using the Applied Biosystems ABI PRISM 7000 Sequence Detector. Hybridization and polymerization were carried out at 60°C for 40 cycles. Quantification was determined using a relative standard curve method using different doses of a reference standard cDNA generated from RNA pooled from Day 81 placentomes. Individual placentome GLUT-1 and GLUT-3 mRNA was expressed relative to the sample's own internal 18S RNA using 18S PDAR kit reagents from Applied Biosystems.

Data Analysis

The physical and endocrine data obtained at autopsy were analyzed by two-way analysis of variance (ANOVA; Minitab 13; Minitab Inc., State College, PA) for the main effect of nutrition and GH treatment and their interaction. Where maternal hormone or glucose concentrations were measured twice weekly, individual means were calculated for the period spanning Days 35–80 and these individual means analyzed by two-way ANOVA as described above. Correlation analyses were by Pearson product- moment test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
One pregnancy in the H + GH group was found to be resorbing at ultrasound examination on Day 75 of gestation, and none of the data pertaining to this pregnancy has been included in the following statistical analyses. Another fetus in the H + GH group was found to have recently died when autopsied on Day 81 of gestation. Fetal size and weight were within the range of the group and the carcass was only mildly autolyzed. Maternal weight and body composition data relating to this pregnancy have been included in the analyses but not the endocrine, fetal, or placental data.

Maternal Dietary Intakes, Live Weight, and Condition Score Changes and Body Composition at Autopsy

Mean weekly maternal feed intakes from embryo transfer to Day 81 of gestation are shown in Figure 1. Maternal dietary intakes were elevated in H compared with M dams throughout this period (P < 0.001) and were largely unaffected by bGH treatment in the M group. There was a modest but significant (P < 0.05) reduction in the voluntary food intake of the H+GH group compared with the H group during the first 2 wk of GH treatment. The marked increases in maternal weight and body condition score in response to high dietary intakes are detailed in Table 1. From an equivalent live weight and adiposity score at the time of embryo transfer, the mean maternal weight gain during the study (corrected for the weight of the gravid uterus) was 47, 43, 215, and 215 g/day for the M, M+GH, H, and H+GH groups, respectively. Thus, bGH treatment did not influence live weight gain in either nutritional group. Similarly, the external subjective assessment of maternal body condition score using the criteria of Russel et al. [23] did not reveal any difference in adiposity due to bGH treatment immediately before autopsy. However, subsequent carcass analysis reveals that both nutrition and bGH treatment had a significant impact on maternal carcass composition (Table 1). High maternal intakes per se significantly influenced the absolute weight, dry matter, crude protein, fat, and ash content of the carcass. The increase in the weight of these components compared with the M-intake group is due primarily to a large increase in fat content. In absolute terms, bGH significantly decreased the dry matter and the fat content of the carcass to different degrees in both M- and H-intake groups. When calculated on a percentage basis, bGH-treated groups had less fat in the maternal carcass (P < 0.003) than untreated groups. Irrespective of treatment group, there was an inverse relationship between the percentage fat and the percentage protein in the maternal carcass (r = –0.914, P < 0.001). The absolute and relative perirenal fat mass of the dams was elevated in the H-intake group compared with the M-intake groups (P < 0.001 and P < 0.05, respectively). Exogenous bGH reduced perirenal fat deposition in both nutritional groups (P < 0.01). Relative maternal liver mass was also elevated in response to H dietary intakes (P < 0.001) and bGH increased liver growth compared with both untreated nutritional groups (P < 0.001).



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  		FIG. 1. Weekly dry-matter food intakes from embryo transfer to autopsy on Day 81 of pregnancy in adolescent dams offered a moderate ({circ}) or high ({square}) intake. Exogenous GH was administered twice daily to an additional moderate (•) or high ({blacksquare}) intake group from Day 35 to 80 of gestation. The period of exogenous GH treatment is indicated by the hatched bar. During Weeks 6 and 7, the voluntary food intakes of the high and high + GH groups were significantly (P < 0.05) different from each other. Values are mean ± SEM; n = 8 per group


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  		TABLE 1. Changes in maternal live weight and body condition score and maternal body composition at Day 81 of gestation in relation to nutrition and exogenous GH treatment.*

Pregnancy Outcome at Autopsy

Uteroplacental parameters and fetal conformation and organ weights at autopsy on Day 81 of gestation are detailed in Table 2. In H-, but not M-intake groups, bGH treatment stimulated (P < 0.001) the growth of the uteroplacenta, as represented by the combined weight of the uterus plus placentomes and fetal membranes. On an individual basis, both the weight of the fetal membranes and the residual uterus in the H+GH group were also statistically different from the other groups. Neither the number of placentomes per placenta nor the mean individual placentome weight was influenced by nutrition or bGH treatment. Exogenous GH treatment in the H-intake group had a profound impact on the combined amniotic and allantochorionic fluid volume, which was increased approximately fourfold relative to the other three groups. At this stage of gestation, the fetus weighs approximately 8–9% of its final birthweight and was not influenced by nutrition or bGH treatment. However, the weights of the fetal liver and kidneys were significantly increased in the H+GH group (P < 0.001 and P < 0.016, respectively). The fetal adrenal glands were also heavier in both M and H groups treated with bGH.


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  		TABLE 2. Uteroplacental parameters and fetal conformation and organ weights at Day 81 of gestation in relation to nutrition and exogenous GH treatment.*

Maternal and Fetal Metabolic and Endocrine Status

Maternal plasma GH concentrations were significantly (P < 0.01) attenuated in H- compared with M-intake dams throughout the study (Fig. 2a). Exogenous bGH treatment, commencing on Day 35 of gestation, stimulated a pharmacological increase in GH during the period of treatment in the M+GH and H+GH dams. Peripheral GH concentrations were higher (P < 0.001) and much more variable in M+GH than in H+GH dams. Maternal insulin concentrations were significantly elevated in H- compared with M- intake dams throughout the study (Fig. 2b). Exogenous bGH treatment stimulated an approximately fivefold increase in peripheral insulin concentrations in both nutritional groups, with the largest relative difference (P < 0.001) in maternal insulin being recorded in the H+GH group. Similarly, maternal glucose concentrations were significantly (P < 0.001) elevated from Day 35 onward in both bGH groups, with the largest relative difference in glucose (a twofold increment relative to the untreated H group) being measured in the H+GH dams (Fig. 2c).



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  		FIG. 2. Changes in maternal (a) GH, (b) insulin, and (c) glucose concentrations, determined throughout the study, in relation to nutrient intake and exogenous GH treatment. Treatments were as follows; moderate intake ({circ}), moderate + GH (•), high intake ({square}), and high + GH ({blacksquare}). Growth hormone was administered twice daily from Day 35 to 80 of gestation as indicated by the hatched bar. Values are mean ± SEM; n = 8 per group

Maternal and fetal plasma hormone and metabolite concentrations immediately preautopsy on Day 81 of gestation are detailed in Table 3. Circulating maternal concentrations of insulin, leptin, and glucose were higher and NEFA lower in H- compared with M-intake dams. Relative to the untreated nutritional groups, bGH administration stimulated higher maternal insulin, IGF-1, and NEFA concentrations, while leptin levels were relatively attenuated. The largest relative differences in maternal IGF-1 and glucose concentrations were recorded in the H+GH group. Maternal alpha- amino nitrogen concentrations were not influenced by nutrition or bGH. In the H+GH group, fetal plasma insulin, glucose, and lactate concentrations were all markedly higher (P < 0.003 to P < 0.001) than in all other groups. IGF- 1 concentrations were lower (P < 0.024) in the fetuses of H-intake dams and not influenced by maternal bGH treatment.


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  		TABLE 3. Maternal and fetal endocrine and metabolic status prior to death on Day 81 of gestation in relation to nutrition and exogenous GH treatment.*

Irrespective of treatment group, the combined amniotic and allantochorionic fluid volume (fetal fluids) was highly associated with preautopsy maternal glucose (r = 0.924, n = 31, P < 0.001), fetal glucose (r = 0.880, P < 0.001), and fetal lactate (r = 0.837, P < 0.001) concentrations (Fig. 3).



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  		FIG. 3. Relationships between (a) maternal glucose, (b) fetal glucose, and (c) fetal lactate concentrations and fetal fluid volume at Day 81 of gestation. Treatments were as follows: moderate intake ({circ}), moderate + GH (•), high intake ({square}), and high + GH ({blacksquare}). Growth hormone was administered twice daily from Day 35 to 80 of gestation

Transplacental Glucose Gradient and Glucose Transporter Expression

The transplacental glucose gradient at autopsy (determined by subtracting the fetal glucose from the maternal glucose concentrations) was significantly influenced by both maternal nutrition and bGH treatment (Table 4). The gradient was on average twofold higher in the H+GH group relative to the other three groups. The expression of GLUT-1 and GLUT-3 relative to 18S RNA was not influenced by nutrition or bGH treatment. However, irrespective of treatment, the ratio of GLUT1mRNA:18S was negatively correlated with the transplacental glucose gradient (r = –0.568, P < 0.01), fetal glucose (r = –0.533, P < 0.01), and maternal glucose (r = –0.481, P < 0.02) concentrations.


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  		TABLE 4. Transplacental glucose gradient and placental GLUT-1 and GLUT-3 mRNA expression in relation to nutrition and exogenous GH treatment.*


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Maternal GH treatment from Day 35 to 80 of pregnancy decreased fat synthesis and increased protein deposition in both H- and M-intake adolescent dams. In H-, but not M- intake groups, this alteration in maternal metabolism was also associated with a significant increase in uteroplacental development and higher fetal liver and kidney weights as determined on Day 81 of gestation. This is the first study to report the effect of exogenous bGH in pregnant ewes during the period of rapid placental proliferation. Furthermore, it is the first study to use singleton-bearing adolescent ewes of two different and known nutritional backgrounds.

The nutritionally induced differences in maternal endocrine and metabolic status, assessed throughout the study or preautopsy for H- compared with M-intake adolescent dams, were identical to those reported and discussed previously. Thus, insulin, IGF-1, glucose, and leptin concentrations were high while fatty acid and GH concentrations were low in the overnourished dams [7, 8, 29]. GH administration significantly increased maternal plasma insulin, IGF-1, glucose, and fatty acid concentrations to different degrees in both nutritional groups. It is well established that GH inhibits tissue responsiveness to insulin, resulting in decreased lipogenesis [33]. In the nonpregnant animal, this would favor redirecting the glucose destined for lipid synthesis to protein (muscle) deposition, but, in the pregnant animal, nutrients can also be partitioned away from maternal stores in favor of the gravid uterus. In this study, maternal insulin and glucose concentrations were twofold higher in response to bGH in overnourished compared with M-intake groups. Thus, the partitioning of nutrients to the pregnant uterus was potentially much greater in the overnourished dams. This may explain why the growth of the uteroplacenta per se was enhanced in the H- but not the M- intake group.

Exogenous GH treatment has been shown to increase uterine horn growth in nonpregnant pigs [34], and the presence of GH receptors in the ovine endometrium and placentome throughout gestation in the sheep [13] suggests that exogenous bGH could also have stimulated uteroplacental growth directly. Exogenous bGH raised maternal GH to extremely high concentrations in both groups, with the highest levels being recorded in the M-intake dams. In spite of this, none of the components that comprise the uteroplacenta were influenced by bGH in the M-intake group, implying that a direct effect of bGH is unlikely in the present study. As detailed in the Introduction, the effect of bGH on uteroplacental growth may be being mediated indirectly via the IGF system in that the various components of the IGF system are expressed by the uteroplacenta throughout gestation. The main site of increased IGF-1 gene expression in GH-treated pregnant pigs is the liver [35], and the highly significant increase in relative maternal liver weight in both M- and H-intake groups in the present study suggests that the threefold increase in maternal IGF-1 concentrations in GH-treated dams reflects increased hepatic IGF-1 synthesis. While it is possible that this IGF-1 influenced placental growth or function via placental IGF-1 receptors, this is perhaps unlikely in view of the lack of effect in the M- compared with the H-intake group. Similarly in rats, maternal IGF-1 administration per se does not influence placental growth [36].

Although maternal GH treatment had a dramatic effect on the growth of the uteroplacenta in the H-intake dams, placental mass and nutrient exchange capacity were clearly not limiting fetal growth at the Day 81 time point, as fetal body weights were similar in all four groups. This is not surprising, as the normally growing fetus has attained only 8% of its final birth weight at this stage, and hence nutrient demand is relatively low. Moreover, the nutritionally mediated placental growth restriction observed during late pregnancy and at term in overnourished or H-intake adolescent dams [3, 21, 37] was not yet evident, at least in terms of placentome and uteroplacental mass. The placentae of the untreated H- compared with M-intake dams were however probably already on a different growth trajectory in that we have recently demonstrated that the proliferative activity of the trophectoderm is attenuated and markers of apoptosis increased in the former group at this gestational timepoint [38].

While fetal body weight was not significantly increased by GH treatment of H-intake dams, the significant increase in fetal liver and kidney weights indicate that these organs were capable of responding to increased nutrient availability during the first half of gestation. Glucose is the primary fetal fuel [39] and the highly significant increase in the transplacental glucose gradient and the resulting high fetal glucose concentrations as determined preautopsy clearly provided an excess of substrate for growth and promoted a hyperinsulinemic state in the H+GH group fetuses. In late pregnancy, fetal insulin is the primary driver of circulating fetal IGF-1 [40]. However, in spite of a fourfold increase in fetal insulin concentrations, maternal GH treatment of H-intake dams did not influence fetal IGF-1 concentrations at Day 81 of gestation. This suggests that the increase in specific fetal organ weights in the H+GH group reflects the increased availability of glucose and implies that, at this relatively early stage of gestation, fetal organ growth is largely independent of the immature fetal endocrine axis. Similarly, increased fetal growth of early gestation pig fetuses in response to maternal GH treatment is independent of changes in fetal plasma IGF-1 or IGF-11 [41]. The positive effects of maternal GH treatment on fetal liver and kidney weights are intriguing in view of the fact that, during late gestation, these organs are the most profoundly perturbed by placental limitation of fetal nutrient supply (J.M. Wallace, D.A. Bourke, and G. Zuur, unpublished data). Thus, it appears that the fetal sheep liver and kidney are highly sensitive to nutrient availability.

The sustained elevation of maternal glucose concentrations in both the GH-treated groups did not influence the placental mRNA expression of the facilitative glucose transporters, GLUT-1 or GLUT-3, although on an individual basis, the expression of GLUT-1mRNA:18S was negatively correlated with the transplacental glucose gradient. In contrast, prolonged maternal hyperglycemia for between 17–20 days in late gestation sheep is associated with marked down-regulation of both placental GLUT-1 and GLUT-3 protein levels [42, 43]. The lack of effect of a much longer period of hyperglycemia on glucose transporter mRNA levels in the present study may be a reflection of the lower expression of both transporters at the earlier stage of gestation being studied (or of posttransciptional control).

As outlined in the Introduction, previous studies in sheep using similar doses of bGH have failed to influence the growth of the uteroplacenta and have had variable effects on fetal weight [12, 16, 17]. However, these studies were carried out in adult ewes with normally growing fetuses and were confounded by litter size. Furthermore, the maximum duration of maternal GH treatment was 14 days. The effect of long-term maternal GH treatment on pregnancy outcome in rapidly growing adolescent animals is as yet unknown, but the results of this initial study suggest that, when fetal nutrient availability is compromised, as becomes evident during the final third of gestation [21, 22], maternal GH treatment may be an effective way of improving fetal nutrient supply.

The major increase in the combined amniotic and allantochorionic fetal fluid volume in response to maternal GH treatment in the H-intake group is of concern in view of the known clinical association between polyhydramnios and fetal death in late pregnancy and at term in humans [44, 45]. The most probable cause in the present study is the 50% increase in fetal lactate concentrations in the hyperglycemic group. Studies in late gestation sheep fetuses show that a modest and sustained artificial elevation in fetal lactate for a period of 3 days results in a 10-fold increase in fetal fluid volume by osmotically drawing fluid from the maternal to the fetal circulation via the placenta [46]. Polyhydramnios has also been widely reported in late gestation sheep and cattle conceptuses cloned by nuclear transfer [47, 48] and in oversized conceptuses produced after in vitro culture of sheep zygotes [49] and may reflect a common etiology based in the original perturbation of the fetal somatotrophic axis.

In conclusion, administration of GH during the period of rapid placental proliferation alters endocrine status in the overnourished adolescent dam and induces a switch from adipose tissue deposition to protein anabolism. This alteration in nutrient partitioning results in enhanced uteroplacental and fetal growth. It remains to be established whether these effects are due wholly to alterations in maternal metabolism or if they also reflect an effect of bGH and/or the IGF system at the level of the uteroplacenta.


   ACKNOWLEDGMENTS
 
We thank Louise Thomas for performing the leptin assay; Terry Atkinson for iodinating insulin, IGF-1, and GH; Neil Leitch for assisting with the autopsies; and Graham Horgan (BIOSS) for statistical advice. The GH radioimmunoassay kit was provided by NIDDKD and the bGH was kindly gifted by Monsanto.


   FOOTNOTES
 
1 Supported by the Scottish Executive Environment and Rural Affairs Department. Back

2 Correspondence: Jacqueline Wallace. FAX: 44 1224 716622; Jacqueline.Wallace{at}rri.sari.ac.uk Back

Received: 23 October 2003.

First decision: 20 November 2003.

Accepted: 23 December 2003.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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