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1 Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK, 1 Department of Physiology, University of Cambridge, Cambridge, UK, 2 School of Medical Sciences, University of Aberdeen, Aberdeen, UK, 3 Health Sciences Center, Perinatal Research Center, University of Colorado, Aurora, Colorado 80010, USA and 4 Colorado State University, Fort Collins, Colorado 80523, USA
Correspondence should be addressed to R G Lea who is now at School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Sutton Bonington LE12 5RD, UK; Email: richard.lea{at}nottingham.ac.uk
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Introduction |
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Human adolescent pregnancies have an increased incidence of IUGR which has been associated with maternal growth and attributed to an endocrine-mediated perturbation in nutrient partitioning between mother and foetus (Scholl & Hediger 1993, Scholl et al. 1997). However, the precise intrauterine mechanisms that underlie IUGR during human adolescent pregnancy are ethically and practically difficult to determine. Although limited progress has been made from the study of human IUGR, the mechanistic basis of IUGR during adolescent pregnancy relies largely on the development of controlled animal models. In a sheep model of human adolescent pregnancy, adolescent ewes carrying singleton pregnancies are offered a normal or high intake (2xmaintenance) designed to maintain maternal adiposity or promote rapid maternal growth respectively. Paradoxically, those animals on the high dietary intake, despite showing increased adiposity, have significantly smaller lambs than the control animals (Wallace et al. 1996, 1997a). Restricted placental growth is central to the pathology of IUGR in this model and has been associated with altered maternal endocrine status including reduced levels of circulating progesterone from as early as day 50 of pregnancy (Wallace et al. 1997a, 1997b, 2001, 2003). These nutrition-driven effects only occur in growing adolescent sheep and foetal IUGR can be partly reversed by supplementation with progesterone from days 5 to 55 (Wallace et al. 2003, 2005a). In addition, the administration of growth hormone (GH), also attenuated in high intake ewes, impacts on uteroplacental growth if given during the period of placental proliferation (Wallace et al. 2004). Interestingly, GH administration later in gestation (days 95–125) does not alter placental mass, but does affect maternal metabolism and induce a modest increase in foetal weight (Wallace et al. 2006a, 2006b). These observations suggest that the hormonal changes are likely mechanistic and important to the process of IUGR, particularly as they occur at mid-gestation prior to changes in placental or foetal size. The hormonal changes are accompanied by the developmental changes at the mid-gestation (day 81) maternal–foetal interface manifest by 1) reduced trophoblast proliferation, 2) increased placental bax protein indicative of apoptotic cell death and 3) reduced expression of several angiogenic growth factors (Lea et al. 2005, Redmer et al. 2005). The relationship between these developmental changes and alterations in placental-derived hormones such as progesterone is uncertain.
In a different sheep model of foetal IUGR where the trigger is exposure of an adult pregnant ewe to hyperthermia, circulating ovine placental lactogen (oPL) concentrations were reduced but placental oPL mRNA levels remained unchanged (Regnault et al. 1999). Consequently, the mechanism was thought to reflect a problem in placental differentiation affecting oPL release. Sheep have a cotyledonary, syneptheliochorial placenta (Fig. 1A
). The foetal trophectoderm layer comprises uninucleate proliferative cells and a population of binucleate trophoblast cells (BNCs; Hoffman & Wooding 1993). These non-proliferative BNCs comprise about 15% of the trophectoderm and are formed from the adjacent uninucleate trophectoderm cells by a process of nuclear division without cytokinesis (Hoffman & Wooding 1993). BNCs become rounded in appearance and form granules containing oPL and other protein products. The BNCs then migrate from the trophectoderm layer and fuse with maternal epithelial cells to form a maternal–foetal hybrid syncytium. Following fusion, the oPL-containing granules translocate to the basal plasmalemma of the syncytium and oPL is released into the maternal compartment by exocytosis (Fig. 1B). Consequently, oPL secretion to the maternal circulation is entirely dependent on BNC migration, which occurs from as early as day 16 of pregnancy (Wooding 1981, Wooding et al. 1986, 1996). BNC migration and fusion is also essential for placental differentiation, so any factors that interfere with this process may also alter placental lactogen secretion.
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Reduced maternal progesterone levels may reflect decreased placental synthesis. At the intracellular level, the rate-limiting step in progesterone synthesis is the conversion of cholesterol to pregnenolone by cytochrome P450scc. This occurs in trophoblast mitochondria and is followed by the conversion of pregnenolone to progesterone by type 1 3ß-hydroxy-steroid dehydrogenase (Strauss et al. 1996). In addition, progesterone synthesis depends on the transport of cholesterol from the outer to the inner mitochondrial membrane where cytochrome P450scc is localised, and this is controlled by the transporter, StAR (reviewed in Tuckey 2005). Once progesterone is produced, it can be converted to androgen (androstenedione) by cytochrome P450c17 that can then be converted to oestrogens by placental aromatase. Consequently, a decrease in placental progesterone production may be associated with increased cytochrome P450c17 (Mason et al. 1989).
In the sheep model of adolescent pregnancy, we investigated 1) potential mechanisms which may underlie reduced maternal levels of progesterone and 2) circulating and placental levels of oPL. We hypothesised that in overnourished adolescent sheep, reduced progesterone may be due to altered placental biosynthesis. Consequently, we have investigated the abundance of RNA transcripts for 1) the cholesterol transporter StAR, 2) the steroidogenic enzymes Cyp11A1 and Hsd3b critical for progesterone synthesis and 3) Cyp17 (encoding cytP450c17) as a possible mediator of progesterone breakdown. Placental oPL transcript abundance and immunoexpression were also measured and compared with the levels measured in the maternal circulation.
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Materials and Methods |
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15% of the total offered (equivalent to ad libitum intakes). Ewes were weighed weekly and their BCS was assessed monthly. The level of feed offered was adjusted thrice each week according to both live weight changes and feed refusals. At 50 days of gestation, pregnancy was confirmed by transabdominal ultrasonography. After day 100 of gestation, the feed intake of the moderate intake group was adjusted weekly to maintain BCS and meet the increasing nutrient demands of the developing foetus. In experiment 1, maternal peripheral progesterone and placental lactogen concentrations were measured in relation to placental and lamb birth weight at term (M: n=6, H: n=7). Other data pertaining to these pregnancies have been published previously (Wallace et al. 1999). In experiment 2, identically treated pregnant ewes from separate studies were killed at days 81 (M: n=11, H: n=13) and 130 of gestation (M: n=21, H: n=22). Other data pertaining to the former study have been published previously (Lea et al. 2005).
Blood sampling and radioimmunoassays
Blood samples were collected from all ewes by jugular venepuncture thrice a week (Monday, Wednesday and Friday) throughout gestation (experiment 1) or immediately prior to necropsy at day 81 or 130 (experiment 2). All samples were taken about 3 h after the morning feed. In experiment 1, plasma oPL concentrations, determined once weekly, were measured in duplicate by a homologous RIA procedure using a highly specific anti-oPL antibody (non-cross-reactive with ovine GH or ovine prolactin) as previously described by Kappes et al.(1992). The assay sensitivity was 100 pg/tube. With a maximum volume of serum at 400 µl, this is equivalent to a sensitivity of 250 pg/ml. We used serum between 10 and 400 µl depending on gestational age and with respect to the individual assay runs described in this paper, consistent detection was observed at 100–150 pg/ml. The intra- and inter-assay coefficients of variation for a high and low plasma quality control were 2.4 and 4.1%, and 4.6 and 5.4% respectively. In experiments 1 and 2, plasma progesterone concentrations, determined thrice weekly or prior to necropsy, were measured in duplicate, as described by Ronayne & Hynes (1990). The assay limit of detection was 0.05 ng/ml and the respective intra- and inter-assay coefficients of variation were 7.5 and 11.4% for experiment 1, and 10.2 and 21.4% for experiment 2.
Tissue collection and fixation
Dams were killed by i.v. administration of an overdose of sodium pentobarbitone (20 ml Euthesate; 200 mg pentobarbitone per millilitre; Willows Francis Veterinary, Crawley, UK) and exsanguination by severing the main blood vessels of the neck. The foetus was killed by intracardiac administration of sodium pentobarbitone (5 ml Euthesate). The umbilical cord was clamped and the foetus dried and weighed. The uteri were dissected and ten representative placentomes removed from the entire length of the gravid uterine horn and weighed. Placentomes were then either frozen at –80 °C for RNA extraction or sliced into 7 mm cross-sections and immersion fixed in 10% neutral buffered formalin for 24 h, followed by 70% ethanol prior to routine wax embedding. All remaining placentomes were weighed in order to calculate the total placentome weight.
Placental RNA isolation
RNA was extracted from frozen whole placentome tissue collected from 21 individual day 81 pregnancies (M: n=11, H: n=10) and 31 individual day 130 pregnancies (M: n=11, H–IUGR: n=8, H+IUGR: n=12). The tissue was pulverised and 50 mg were added to 0.5 ml lysis buffer containing guanidine isothiocyanate and 2-mercaptoethanol (Sigma lysis solution). The sample was then homogenised and RNA extracted according to the protocol supplied with the GenElute Mammalian Total RNA extraction kit (Sigma-Aldrich Inc.). The quality and quantity of total RNA were determined via capillary electrophoresis using an Agilent 2100 Bioanalyzer (Agilent technologies, Wilmingham, DE, USA).
RT and real-time PCR
All reagents and procedures used for real-time RT-PCR were purchased from and used as directed by Applied Biosystems (Warrington, Cheshire, UK). Real-time PCR was performed using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). For each sample, 30 ng total RNA was reverse transcribed in triplicate using TaqMan RT reagents and multiscribe reverse transcriptase. Ovine primer sequences (Table 1) were obtained either directly through collaboration (Hsd3b) from the literature (oPL, Cyp11A1) or by slight modification of published/available sequences from other species (StAR and cyclophilin respectively). Real-time RT-PCR was carried out using the SYBR green I nucleic acid stain, and the polymerisation and amplification reactions were carried out in triplicate using 96-well PCR plates. Five microlitres of cDNA were added to 20 µl Master Mix and a final volume of 10 µl added to the real-time plate. The plate was incubated in an ABI PRISM 7700 Sequence Detector (Applied Biosystems) under the following conditions: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Quantification was determined using a relative standard curve method with different doses of a reference standard cDNA generated from RNA pooled from eight day 81 placentomes (4xM+4xH) or eight day 130 placentomes (4xM+4xH). Individual placentome mRNA for each gene of interest was expressed relative to the housekeeping gene cyclophilin, which remained unaffected by treatment/group in subsequent studies. Primer concentrations were optimised prior to analysis (Table 1) and primer–dimer formation was excluded by running a melt curve for each primer pair. The mean coefficients of variation were oPL (6.7%), StAR (9.6%), Cyp11A1 (16.5%), Hsd3b (9.2%), Cyp17 (11.9%) and cyclophilin (16.9%).
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The oPL staining was quantified by computer-aided image analysis using a Zeiss Axioplan microscope (20x objective; Carl Zeiss Ltd, Welwyn Garden City, Herts, UK) and Hitachi HV-C20 colour camera (Hitashi Denshi, Tokyo, Japan) connected to a computer running Image Pro Plus software (Media Cybernetics, Silver Spring, MD, USA). Quantification was carried out over ten fields of view taken randomly and transversely across the central part of the placentome section (200x magnification=4.2x105 µm2; Fig. 3). On each captured image, the total number of positively stained areas (brown colour) was obtained as well as the percentage area stained. As an initial quality control step, brown-stained cells and regions were manually counted in ten fields of view from a single placentome. This was carried out by two independent observers (S M and K W). The image analysis system was then used to count the same areas and the results were found to be comparable (P<0.001, R2=0.83, n=10). Since some staining was observed in syncytial segments, the mean percentage area stained values were used for statistical analysis.
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Effects of nutrition on maternal oPL concentrations across gestation
Maternal plasma oPL concentrations throughout gestation are illustrated in Fig. 2A. Compared with moderately fed controls (M: n=6), animals on the high nutrient intake diet (H: n=7) exhibited lower levels of plasma oPL across gestation with levels significantly diverging at day 51 (P<0.05) and for the remainder of pregnancy (days 58–105: 21.5±4.0 vs 7.6±0.7 ng/ml, P<0.003; days 111–146: 83.4±16.1 vs 25.1±4.3 ng/ml, P<0.02; Student’s t-test on log-transformed data). oPL was also detected later in the high intake group than in the controls. For example, H group animals exhibited a 7-day delay in the rise of plasma oPL above a threshold of 0.5 ng/ml (55.5±1.5 vs 48.0±1.86 days; P<0.01 by Student’s t-test).
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Effects of nutrition on maternal progesterone across gestation
Figure 2B
shows maternal plasma progesterone concentrations determined from blood samples taken thrice a week until term. Mean ovulation rates at embryo transfer were 2.0±0.5 and 2.7±0.4 corpora lutea for the moderate (n=6) and high intake (n=7) animals respectively, and there was no significant difference between the groups. During the first third of pregnancy (13–48 days), overall mean progesterone concentrations were not significantly different between M and H animals. However, the mean progesterone concentrations for the middle and final thirds of gestation were significantly lower in H intake animals than in M controls (days 51–105: 5.1±0.3 vs 10.0±1.3 ng/ml, P<0.01; days 108–138: 4.5±0.7 vs 15.8±2.8 ng/ml: P<0.01; Student’s t-test on log-transformed data).
In the first third of gestation (13–48 days), there were no significant correlations between maternal plasma progesterone concentrations and placental weight, cotyledon weight or foetal weight. Regression analysis of log-transformed mean progesterone concentrations across the middle third of gestation (days 51–105) after adjusting for diet effects revealed a significant positive association with placental weight (P<0.05, R2=0.62, n=13) and total foetal cotyledon weight (P<0.05, R2=0.77, n=13). Across the final third of gestation (days 108–138), log plasma progesterone concentrations positively associated with placental weight (P<0.05, R2=0.45, n=13), total foetal cotyledon weight (P<0.05, R2=0.58, n=13) and foetal weight (P<0.01, R2=0.51, n=13).
During the first third of gestation, there was no significant association between maternal progesterone and oPL concentrations. However, during the middle third of gestation (progesterone: 51–105 days vs oPL: 58–105 days), progesterone was significantly and positively associated with oPL after adjusting for diet (P<0.05, R2=0.63, n=13). During the final third of gestation, a significant relationship was suggested since the P value fell just outside the 5% level (P=0.056, R2=0.65, n=13).
Experiment 2
Maternal live weight and body condition
Embryo transfers carried out on days 81 and 130 were separate studies. In the day 81 study, at embryo transfer, the mean live weight and BCSs were 45±0.8 vs 46± 0.9 kg and 2.3±0.04 vs 2.4±0.04 score units in the H (n=13) and M (n=11) group respectively. In the day 130 group, at embryo transfer, the mean live weight and BCSs were 44±0.8 vs 46±0.8 kg and 2.3±0.03 vs 2.3±0.04 score units in the H (n=22) and M (n=21) groups respectively. The diet-induced changes in maternal weight and BCS in day 81 and 130 cohorts in relation to placental and foetal weight at necropsy are shown in Table 2. Mean maternal live weight gain (g/day) and BCSs were significantly increased in the ad libitum fed H group relative to the M intake control group (P<0.001) as determined at both gestational time-points.
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). Fourteen foetuses were male in both the H and the M groups at this time-point. The late gestation H group was further divided into ewes with IUGR foetuses (H+IUGR) and less perturbed animals (H–IUGR). By one-way ANOVA, placental and foetal size was significantly affected across the three groups (P<0.0001). The H+IUGR group (n=14) had significantly smaller placentas than the H–IUGR group (n=8; P<0.01, post hoc t-test) and the control M group animals (n=21; P<0.0001, post hoc t-test; 216.29± 18.99, 356.38±43.99 and 458.24±28.69 g respectively). Similarly, foetal weight was significantly reduced in the H+IUGR group when compared with the H–IUGR group (P<0.001, post hoc t-test) and the control M animals (P<0.0001; post hoc t-test; 2900.50±203.06, 4181.38±157.06 and 4525.71± 90.83 g respectively). As previously reported (Wallace et al. 2000, 2002, 2006b), the IUGR observed was asymmetric since foetal brain/liver weight ratios were significantly affected across the three late gestation groups (P<0.001, one-way ANOVA) and significantly increased in the IUGR group (M vs H–IUGR vs H+IUGR: 0.32±0.01 vs 0.34±0.02 vs 0.51±0.05 respectively, P<0.05; post hoc t-test). The mean maternal live weight gain of the H+IUGR group was not significantly different from that of the H–IUGR group (282.24±16.85 and 272.14±10.57 g/day respectively), and adiposity scores at necropsy were equivalent (3.3±0.13 and 3.3±0.08 score units respectively).
Effects of nutrition on placental oPL mRNA
Placental oPL mRNA was confirmed by real-time PCR using ovine-specific primers (Table 1). Comparison of M and H groups at days 81 and 130 of gestation showed no significant effect of nutrition on placental oPL transcript abundance when expressed relative to cyclophilin (day 81 M vs H: 0.79±0.09 vs 0.61±0.08 respectively; day 130 M vs H–IUGR vs H+IUGR: 1.93±0.10 vs 2.00± 0.23 vs 1.77±0.17 respectively). Similarly, there were no significant differences in the transcript abundance of the gene per se (analysed independently; data not shown) and the abundance of cyclophilin transcripts was not affected by treatment at either day 81 or 130 (data not shown).
Effects of nutrition on placental oPL protein
In all placental samples examined, oPL was localised to the binuclear cell population at the maternal foetal interface and, particularly evident in late gestation, to areas of syncytium where BNC fusion had recently occurred (Fig. 3A, day 81; 3B, day 130). There were no significant differences attributed to maternal nutritional treatment in the placental oPL protein expression at either day 81 (M vs H: 3.2±0.5 vs 2.6±0.3) or day 130 (M vs H: 6.6±1.0 vs 5.4±4.2). This remained consistent following subdivision of the late gestation H animals into H–IUGR and H+IUGR subgroups (5.4±1.5 vs 5.4± 1.1 respectively). At late gestation, placental oPL staining was positively associated with placental weight when all groups were analysed together (P<0.01, R2=0.20, n=43), in the moderate group (P<0.05, R2=0.22, n=21) and in the combined H group (P<0.05, R2=0.17, n=22). There was no correlation between oPL protein and foetal weight in any group.
Effects of nutrition on maternal progesterone
As presented in detail for experiment 1, maternal peripheral progesterone concentrations were significantly reduced at mid- and late gestation in H when compared with M dams (P<0.01 and P<0.001 respectively; Table 3). In the late gestation cohort, subdivision of the H group on the basis of foetal weight revealed that maternal progesterone was significantly affected across the three late gestation groups (P<0.0001; one-way ANOVA) and was significantly lower in those animals with IUGR (H+IUGR) when compared with the M (P<0.0001: post hoc t-test) and H–IUGR (P<0.01) groups (Fig. 4).
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Transcript abundance of StAR, Cyp11A1, Hsd3b and Cyp17 in mid- (day 81) and late gestation (day 130) placentomes
Placental RNA transcripts for StAR, Cyp11A1, Hsd3b and Cyp17 were detected in both mid- and late gestation placental samples. At day 81, there was no significant difference between the M and the H groups for StAR or any of the steroidogenic enzyme mRNA measured (M vs H for StAR: 0.47±0.06 vs 0.46±0.05, Cyp11A1: 4.89±0.68 vs 6.48±0.61, Hsd3b: 0.63±0.04 vs 0.74±0.09, Cyp17: 0.66±0.08 vs 0.84±0.18). Placental transcript abundance for Cyp11A1 was significantly affected across the three late gestation groups (P<0.05; one-way ANOVA) and was lower in the H+IUGR group when compared with the M (P<0.01: post hoc t-test) and the H–IUGR (P<0.01) groups (Table 4). Late gestation placental mRNAs for StAR, Hsd3b and Cyp17 were independent of nutrition and foetal growth status. Data expressed relative to the control cyclophilin gene and calculated independently (data not shown) showed similar results.
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Data presented here illustrate that despite the dramatic reduction in systemic oPL in response to altered maternal nutrition, there is no accompanying reduction in placental oPL mRNA or protein at day 81 or 130 of gestation. Similarly, with respect to progesterone, there was no alteration in StAR or the steroidogenic enzymes at day 81. However, at day 130, the most perturbed H intake ewes with IUGR showed a reduction in the abundance of Cyp11A1 transcripts. In contrast, StAR and Hsd3b were unaffected as was the expression of Cyp17 that converts progesterone to androstenedione. These data suggest that at mid-gestation, reduced concentrations of oPL and progesterone in the peripheral maternal circulation do not reflect changes in placental oPL expression or the expression of StAR and the steroidogenic enzymes necessary for progesterone synthesis. Moreover, these endocrine changes do not reflect placental size since this is equivalent in both groups at this mid-gestation time-point. We propose therefore that the mid-gestation reduction in maternal oPL may reflect altered BNC trophoblast fusion and/or the altered control of granule exocytosis subsequently affecting hormone release. With respect to the mid-gestation reduction in progesterone, we postulate that this likely reflects increased maternal metabolic clearance by the liver. However, in late gestation, reduced maternal progesterone in animals with IUGR partly reflects reduced placental size, but is also associated with reduced progesterone biosynthesis due to decreased Cyp11A1 mRNA. Although StAR and Hsd3b were not altered in this study, they were expressed in all placentas examined thus confirming previous reports (Su et al. 2004, Vaneslow et al. 2004). The lack of an effect of nutrition on placental Cyp17 indicates that increased progesterone breakdown is an unlikely explanation for reduced circulating steroid.
Reduced circulating oPL concentrations with no change in placental oPL mRNA or protein have been previously reported in a sheep model of placental insufficiency and foetal IUGR induced by maternal hyperthermia (Regnault et al. 1999). In this model, pregnant adult ewes were exposed to high ambient temperatures during the first and second thirds of gestation (Bell et al. 1987, Thureen et al. 1992) and, similar to the adolescent model, both oPL and progesterone concentrations were reduced at mid-gestation (day 93) with no significant reduction in placental size (Regnault et al. 1999). In contrast to the adolescent model, foetal size was reduced at day 93, and this is thought to reflect the greater severity of hyperthermia, when compared with altered nutrition, as a stimulus for IUGR (Wallace et al. 2005b). Notwithstanding, it is possible that the mechanism of reduced circulating oPL levels may be the same in both models. Indeed, the present study used the same RIA as the hyperthermia model and therefore both studies used the same highly specific and non-cross-reactive anti-oPL antibody (Kappes et al. 1992). With respect to the importance of oPL for foetal development, there is evidence that placental lactogen infused directly into the late gestation ovine foetus has subtle effects on some major organ systems (Schoknecht et al. 1996). However, it is recognised that a direct role in the induction of foetal IUGR requires further investigation.
Placental oPL release into the maternal circulation depends on the migration of BNCs and fusion with the maternal epithelium. However, the total amount detectable in the circulation will also be influenced by placental mass. In experiment 1, late gestation maternal oPL concentrations were reduced by 69% (days 111–146), whereas term placental size was reduced by 43%. Therefore, it follows that the reduced concentration of oPL in the maternal circulation likely reflects both reduced placental mass and impaired trophoblast migration subsequently affecting hormone release following BNC fusion. In addition to lower levels of oPL in high nutrient intake animals, the gestational rise above a minimal threshold is delayed by at least 7 days. The reason for this is likely developmental and may also relate to inadequate BNC migration and fusion. In support of these possibilities, day 80 pregnant adolescent sheep on high nutrient intake diets exhibit reduced proliferative activity of the foetal trophectoderm accompanied by an increased expression of the pro-apoptotic protein bax (Lea et al. 2005). This indicates an altered proliferation/ apoptosis balance in the placenta which could affect trophoblast cell migration and fusion. Interestingly, pregnancy-specific protein B (PSPB) is also released from BNC granules and shows similar changes to oPL in response to high intake in the adolescent model (Wallace et al. 1997b, Green et al. 2000). Therefore, it follows that PSPB may be similarly affected by BNC migration and fusion.
In contrast to oPL release, progesterone secretion occurs by diffusion from ovine trophoblast cells. Enriched BNCs have been shown to synthesise progesterone from pregnenolone and uninucleate trophoblast plus the syncytium are reported to be immunopositive for 3ß-hydroxysteroid dehydrogenase essential for progesterone biosynthesis (Riley et al. 1991, Wango et al. 1991). Consequently, progesterone will be released into both the foetal and the maternal circulations from trophoblast cells.
During ovine pregnancy, the placenta becomes the predominant source of progesterone from day 50 of gestation (Al-Gubory et al. 1999). Indeed, although the corpus luteum in the ewe persists until near term, it has been calculated that the late gestation placenta produces five times as much progesterone as the ovary (Linzell & Heap 1968). Since there was no nutritional effect on circulating progesterone during the first 50 days of pregnancy, i.e. prior to the luteo-placental shift, it is likely that the 50% decline seen in late gestation is attributable to a dietary effect on placental rather than corpus luteum-derived progesterone. Notwithstanding, a modest effect of diet on ovarian progesterone cannot be ruled out. The rate-determining step in the synthesis of progesterone is the conversion of cholesterol to pregnenolone by cytochrome P450scc (Tuckey 2005). Consequently, reduced progesterone in the late gestation high nutrient intake group with IUGR may be consequential to reduced Cyp11A1 encoding cytochrome P450scc in the same subgroup. However, since Cyp11A1 was not altered earlier in gestation, despite reduced circulating levels of progesterone, the reduction in late gestation is likely a consequence of rather than a cause of IUGR. Therefore, it follows that these changes must follow events occurring earlier in gestation. In support of this, mid-gestation alterations in the placental expression of angiogenic factors have been linked to a reduction in absolute uteroplacental blood flow in late gestation (Wallace et al. 2002, Redmer et al. 2005). Since, in the human, an increase in progesterone production during pregnancy has been associated with an increased blood supply to the placenta (reviewed in Tuckey 2005), altered placental endocrine function in the overnourished adolescent sheep may partially reflect developmental/vascular abnormalities occurring earlier in gestation.
Increased feed intake in non-pregnant adult sheep decreases circulating progesterone and increases blood flow in the liver and hepatic portal vein (Burrin et al. 1989, Parr 1992, Parr et al. 1993). The liver is the major site of progesterone inactivation and Parr et al.(1993) demonstrated that increasing feed intake in non-pregnant sheep lowers circulating progesterone by increasing the metabolic clearance rate. Similarly, in a separate study on lactating and non-lactating cows, both liver blood flow and steroid clearance were affected by feed intake (Sangsritavong et al. 2002). Increased dietary intake during pregnancy also appears to increase steroid clearance. Pregnant adult sheep offered the same high quantities of the complete diet as the adolescent animals in the present study exhibit reduced progesterone concentrations but, when compared with controls, there was no significant difference in placental and foetal weights (Wallace et al. 2005a). Recent studies in overnourished adolescent sheep have shown that a 50% reduction in circulating progesterone in late gestation (day 130) can be reversed by switching animals from a high to a low intake diet at day 90 (moderate: 13.9±1.0; high: 6.9±0.83; high to low: 17.3±1.44). This change in peripheral progesterone levels occurred independent of changes in placental size in that equivalent placental growth restriction was evident in high and high–low groups (293±23.9 vs 375±48.0 g respectively; D A Redmer and J M Wallace, unpublished observations). In contrast to the overfeeding model, underfeeding adolescent ewes (0.75% maintenance diet from embryo transfer) has no effect on term placental size but increases circulating progesterone concentrations by 70% (J S Luther and J M Wallace, unpublished observations). In these experiments, switching the animals to a moderate intake diet at day 90 decreased circulating progesterone to levels comparable with the moderate intake control group by day 130 (moderate: 18.8±3.4; underfed: 32.1±5.0; underfed to moderate: 21.2±2.9). In the present study, mid-gestation day 81 ewes on a high intake diet exhibit a decrease in circulating progesterone but no difference in placental size. Since the live weight gain averaged to day 81 is significantly greater in the overnourished group, these animals have a higher metabolic activity. Indeed, a sustained elevation of maternal tri-iodothyronine and thyroxine concentrations in high intake adolescent ewes from mid-gestation provides further evidence of an increase in metabolic rate (Wallace et al. 1997a). Therefore, it is likely that the mid-gestation decrease in circulating progesterone is due to an increase in maternal metabolic clearance.
At late gestation, ewes on high nutrient intakes, both with and without IUGR foetuses, had a higher mean live weight gain than moderately fed control ewes. Therefore, an increase in maternal metabolic clearance may at least partly account for lower levels of progesterone in late gestation high intake ewes. However, progesterone concentrations in the most perturbed high intake ewes with IUGR foetuses were 56% lower than in high intake ewes with non-IUGR foetuses. Since the mean live weight gain of the two high intake groups was equivalent, maternal metabolic clearance clearly does not entirely account for the decrease in progesterone observed in IUGR pregnancies. Low maternal progesterone levels in this subgroup appear to be associated with the smaller placental size that characterises this group. In addition, the decreased abundance of Cyp11A1 transcripts suggests that the smaller placentas also exhibit reduced biosynthetic activity and are unable to compensate for the reduced size. In support of a possible contributory effect on biosynthesis, we have shown that late gestation peripheral progesterone concentrations positively associate with the expression of both Cyp11A1 and Hsd3b, whereas there was no evidence for a similar relationship at mid-gestation.
In conclusion, mid- and late gestation overnourished adolescent sheep exhibit reduced systemic levels of oPL and progesterone. At mid-gestation, there are no similar changes in the expression of placental oPL, StAR and the steroidogenic enzymes important for progesterone synthesis. Since placental size is equivalent at this time point, mid-gestation circulating maternal levels of oPL and progesterone most likely reflect altered placental differentiation and/or, with respect to progesterone, increased maternal metabolic clearance. At late gestation, the lowest progesterone concentrations were found in the high intake animals with IUGR. These ewes also had smaller placentas than non-IUGR high intake animals and a decreased expression of placental Cyp11A1 indicative of altered progesterone biosynthesis. Since the high intake subgroups had equivocal live weight gains, reduced placental size and impaired progesterone biosynthesis appear to be key contributory factors to the late gestation reduction in circulating progesterone in IUGR pregnancies.
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References |
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). Progesterone was measured thrice weekly and oPL once weekly across gestation.