Maternal Undernutrition from Early- to Mid-Gestation Leads ... - BioOne

BIOLOGY OF REPRODUCTION 69, 133–140 (2003) Published online before print 19 February 2003. DOI 10.1095/biolreprod.102.012120

Maternal Undernutrition from Early- to Mid-Gestation Leads to Growth Retardation, Cardiac Ventricular Hypertrophy, and Increased Liver Weight in the Fetal Sheep Kimberly A. Vonnahme,2 Bret W. Hess,2 Thomas R. Hansen,2 Richard J. McCormick,2 Daniel C. Rule,2 Gary E. Moss,2 William J. Murdoch,2 Mark J. Nijland,4 Donal C. Skinner,3 Peter W. Nathanielsz,4 and Stephen P. Ford1,2 Department of Animal Science2 and Department of Zoology,3 University of Wyoming, Laramie, Wyoming 82071-3684 Department of Obstetrics and Gynecology,4 New York University School of Medicine, New York, New York 10016 ABSTRACT

considerably even when genetics and nutritional management are constant. These differences in animal performance and composition have been attributed to variations in both endogenous and exogenous hormonal status [4] and to the general health and immune status [3] of the animal. Epidemiological studies in humans have revealed that undernutrition during the first half of pregnancy alters fetal growth and development, predisposing offspring to cardiovascular, metabolic, and endocrine diseases in adult life [5– 8]. In sheep, as well as humans, the first half of gestation is critical for proper fetal organogenesis and placental growth and vascularization [9–11]. Further, the structure of fetal blood vessels (stem arteries and veins, intermediate arterioles and venules, and terminal capillaries) in sheep and humans are comparable [12], suggesting similarities in fetal placental vascular development. Vascular endothelial growth factor (VEGF), a potent angiogenic [13] and permeability [14] factor, is present in the cotyledon of the ovine placentome [15] and is regulated by hypoxia [16] and estrogen [17]. It is probable that undernutrition in the dam may impact placental vascularity and the transport of nutrients and oxygen to the growing fetus. Although several investigations [18–20] have looked at a moderate and decreasing nutrient restriction during gestation in the ewe, none resulted in significant losses in maternal body weight or fetal weight during the restriction period. Further, none of these studies evaluated the impact of undernutrition on placentomal (cotyledonary/caruncular) vascularity. We hypothesized that a constant reduction in nutrient intake to 50% NRC [21] requirements from Days 28 through 78 of gestation would decrease maternal body weight and reduce the growth and development of the ovine fetus. More specifically, the objectives of this study were to analyze the impact of a constant nutrient restriction on maternal and fetal body weights and selected organ weights, as well as placentomal numbers, weights, morphologies, and vascularities. In addition, the impact of nutrient restriction on levels of glucose, tri-iodothyronine (T3) and thyroxine (T4), estradiol-17b (E2-17B), and progesterone (P4) in maternal and fetal blood and vascular endothelial growth factor (VEGF) in fetal blood were evaluated.

Early gestation is critical for placentomal growth, differentiation, and vascularization, as well as fetal organogenesis. The fetal origins of adult disease hypothesis proposes that alterations in fetal nutrition and endocrine status result in developmental adaptations that permanently change structure, physiology, and metabolism, thereby predisposing individuals to cardiovascular, metabolic, and endocrine disease in adult life. Multiparous ewes were fed to 50% (nutrient restricted) or 100% (control fed) of total digestible nutrients from Days 28 to 78 of gestation. All ewes were weighed weekly and diets adjusted for individual weight loss or gain. Ewes were killed on Day 78 of gestation and gravid uteri recovered. Fetal body and organ weights were determined, and numbers, morphologies, diameters, and weights of all placentomes were obtained. From Day 28 to Day 78, restricted ewes lost 7.4% of body weight, while control ewes gained 7.5%. Maternal and fetal blood glucose concentrations were reduced in restricted versus control pregnancies. Fetuses were markedly smaller in the restricted group than in the control group. Further, restricted fetuses exhibited greater rightand left-ventricular and liver weights per unit fetal weight than control fetuses. No treatment differences were observed in any gross placentomal measurement. However, caruncular vascularity was enhanced in conceptuses from nutrient-restricted ewes but only in twin pregnancies. While these alterations in fetal/ placental development may be beneficial to early fetal survival in the face of a nutrient restriction, their effects later in gestation as well as in postnatal life need further investigation.

conceptus, placenta, placental transport, pregnancy, uterus

INTRODUCTION

Climates with extreme variations in precipitation routinely experience significant fluctuations on both quantity and quality of forage [1, 2]. In fact, prolonged bouts where .50% of the National Research Council’s (NRC) requirements for gestation are not met are common [3]. Thomas and Kott [3] concluded that unsupplemented ewes on rangeland lose a significant amount of weight from early to mid-gestation and that, even after supplementation later in gestation, the health of their lambs is compromised. Growth and carcass characteristics of ruminants vary

MATERIALS AND METHODS

Correspondence: Stephen P. Ford, 113 Animal Science and Molecular Biology Complex, University of Wyoming, Laramie, WY 82071-3684. FAX: 307 766 2355; e-mail: [email protected]

1

Animals All animal procedures were approved by the University of Wyoming Animal Care and Use Committee. On Day 20 of pregnancy, 13 multiparous ewes of mixed breeding were weighed so that individual diets could be calculated on a metabolic body weight basis (weight0.75). The diet consisted of a pelleted beet pulp (79.7% total digestible nutrients [TDN], 93.5% dry matter [DM], and 10.0% crude protein). Rations were delivered

Received: 10 October 2002. First decision: 8 November 2002. Accepted: 11 February 2003. Q 2003 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

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FIG. 1. A) Cross section of a Day 78 type A ovine placentome. B) 340 magnification of the maternal:fetal interface. Cot, cotyledonary tissue; Car, caruncular tissue; *, cotyledonary blood vessels; **, caruncular blood vessels.

on a DM basis to meet the total TDN required for maintenance for an early pregnant ewe (NRC requirements [21]). A mineral-vitamin mixture (51.43% sodium triphosphate, 47.62% potassium chloride, 0.39% zinc oxide, 0.06% cobalt acetate, and 0.50% ADE vitamin premix [8 000 000 IU vitamin A, 800 000 IU vitamin D3, and 400 000 IU vitamin E per pound; amount of vitamin premix was formulated to meet the vitamin A requirements]) was included with the beet pulp pellets to meet requirements. On Day 21 of gestation, all ewes were placed in individual pens and fed control rations. On Day 28, ewes were randomly assigned to a control-fed group (n 5 7; 100% NRC requirements [21], which included 100% mineral-vitamin mixture) and a nutrient-restricted group (n 5 6; fed 50% NRC requirements [21], which included 50% mineral-vitamin mixture). Beginning on Day 28 of gestation and continuing at 7-day intervals, ewes were weighed and rations adjusted for weight gain (i.e., increased the amount of feed) or loss (i.e., decreased the amount of feed). On Day 45 of gestation, the numbers of fetuses carried by each ewe was determined by ultrasonography (Ausonics Microimager 1000 sector scanning instrument; Ausonics Pty Ltd, Sydney, Australia). Just prior to ewes being killed on Day 78 of gestation, each ewe was weighed and a sample of blood collected via jugular venipuncture into a heparinized vacutainer tube (sodium heparin, 143 USP units, Becton Dickinson, Franklin Lakes, NJ) for T3, T4, P4, and E2-17B determination and into a separate tube (heparin plus sodium fluoride; 2.5 mg/ml; Sigma, St. Louis, MO) for glucose determination. Ewes were then given an overdose of sodium pentabarbitol (Abbott Laboratories, Abbott Park, IL) and exanguinated, and the gravid uterus was quickly removed and weighed. The tip of the gravid uterine horn was opened to expose the fetus, and the umbilical cord was located. Umbilical cord blood was collected into a heparinized vacutainer tube for T3, T4, P4, E2-17B, and VEGF and a tube containing heparin plus sodium fluoride for glucose determination. Fetal weights, crown-rump lengths, abdominal circumferences and sex, and the weights of the liver, pancreas, lung, kidney, adrenal, and the left ventricle, right ventricle, and septum of the heart were recorded. Maternal and fetal blood were collected into cooled tubes and placed on ice and centrifuged (48C, 3000 g, 10 min). Maternal and fetal plasma were stored at 2808C until analyzed. After removal of the fetus, a type A placentome [22] located ;10 cm from the umbilicus was selected, dissected from the surrounding tissues

and weighed. A cross section of the placentome was placed in a tissue cassette (Tissue Tek, Miles Labs, Elkhart, IN) and fixed with paraformaldehyde and paraffin embedded. Twelve 5-mm sections evenly spaced over a 450-mm area of each placentome were evaluated for vascular density via image analysis [23] (Optimus Image Analysis Software, Bothell, WA). Briefly, maternal and fetal blood vessels were counted and traced within four fields per section at points where the caruncular and adjacent cotyledonary tissue of the placentome could be visualized (Fig. 1). The blood vessel area per unit tissue area (i.e., caruncular blood vessel area/caruncular area; cotyledonary blood vessel area/cotyledonary area), number of blood vessels per unit tissue area (i.e., caruncular blood vessel number/ caruncular area; cotyledonary blood vessel number/cotyledonary area), and blood vessel diameters were calculated. All remaining placentomes were removed and weighed, and their diameter and morphologic type (based on the classification scheme of Vatnick et al. [22]) were recorded. By using the diameter measurements previously recorded, the surface area of each placentome was calculated by using the formula pr2 (r 5 radius). Thereafter, the cotyledonary tissue was peeled away from the caruncular tissue of each placentome and the components from all placentomes pooled so that total caruncular and total cotyledonary weights could be determined. Maternal organs including the pituitary gland, adrenal gland, liver, and left ventricle, right ventricle, and septum of the heart were removed from the body cavity and weighed. The reticulo-rumen, omasum, abomasum, and entire gastrointestinal tract were emptied of their contents and weighed, and maternal empty body weights were recorded.

Assays Glucose was analyzed using a colorimetric assay according to manufacturer’s specifications (Sigma, Glucose catalog no. 115-A). Within-assay variability for glucose was determined by assaying a high and low pool of systemic plasma from pregnant ewes. The intraassay and interassay (n 5 7) coefficients of variation (CV) were 4.5% and 13.5% versus 13.1% and 19.4% versus for the high and low pools, respectively. Estradiol-17b was quantified in 1-ml maternal and fetal plasma samples. Extraction, chromatography, and radioimmunoassay (RIA) procedures were identical to procedures previously validated in this laboratory

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UNDERNUTRITION AND FETAL COMPENSATION

TABLE 1. Maternal organ weights on Day 78 of gestation from controlfed (n 5 7) and nutrient-restricted (n 5 6) ewes. Organ weight Gravid uterus, kg Pituitary, g Adrenal, g Left ventricle, g Right ventricle, g Septum, g Liver, g Total intestine, g Reticulo-rumen, g Omasum, g Abomasum, g Colon, g Cecum, g a

FIG. 2. Changes in body weight of control-fed (solid symbols) and nutrient-restricted ewes (open symbols) during the experimental period.

for ovine plasma and using the same fully characterized antibody [24]. All samples were analyzed in the same assay; intraassay CV was ,10%. Progesterone was measured via a specific RIA previously validated in our laboratory for pregnant ewe plasma [25] utilizing the same fully characterized antibody, GDN-337 [26]. All samples for P4 determination were run in a single assay (intraassay CV 5 7.1%). Tri-iodothyronine and T4 were determined by RIA according to manufacturer’s specifications (Coata-Count Total T3 and T4, DPC, Los Angeles, CA). All blood plasma samples were assayed in a single assay. The intraassay CV was 8.8% and 7.7% for T3 and T4, respectively. An RIA for VEGF was developed for sheep plasma samples according to the protocol of Anthony et al. [27] with modifications. Human, recombinant VEGF165 (cold hormone; G143AB; Genentech, Inc., Los Angeles, CA), primary antibody (polyclonal rabbit antiserum to VEGF165; #27906– 17, Genentech, Inc., Los Angeles, CA) and human, recombinant [125I]VEGF165 (tracer; NEX328, NEN Life Science Products, Inc., Boston, MA) were used in all assays. The 165-isoform of VEGF exists as a soluble form [28] and is the most abundant isoform expressed in ovine placental tissues [15]. Sensitivity averaged 25 pg/ml, defined as the VEGF standard yielding 95% of the counts in the buffer control tube. Within-assay variability for VEGF was determined by assaying a pool of systemic plasma from a pregnant ewe to which known quantities of VEGF had been added (0.0, 0.5, and 5.0 ng/ml plasma). The resulting concentrations (6SEM), after subtraction of the plasma blank (1.46 6 0.11 ng/ml), averaged 0.62 6 0.04 (n 5 4) and 5.42 6 0.16 (n 5 4) ng/ml, respectively. Coefficients of variation averaged 10.7%, 8.2%, and 5.8% for the plasma blank and 0.5- and 5.0-ng/ml VEGF additions, respectively. Parallelism was obtained between a doubly diluted pregnant plasma pool and the standard curve. No cross-reactivity was found with basic fibroblast growth factor or a2macroglobulin (Sigma) at concentrations as high as 100 mg/L.

Statistics Data were analyzed by a factorial analysis of variance using the PROC GLM and PROC CORR procedures of SAS (SAS Institute Inc., Cary, NC). There was no effect of sex on any of the measurements taken; therefore, data were pooled across sex. Class statements included diet and number of fetuses. Model statements included the effects of diet and fetal number on fetal weight, fetal crown-rump length, fetal organ weights, fetal organ weights divided by the weight of the fetus, placentomal number, placentomal surface area, placentomal weight, total caruncular weight, total cotyledonary weight, diameters of blood vessels in caruncular and cotyledonary tissues, blood vessel diameters, blood vessel area per unit area caruncular and cotyledonary tissue and numbers of blood vessels per unit area caruncular and cotyledonary tissues, progesterone, glucose, T3, T4, and VEGF. Means separation was performed using LSMEANS. Correlations between these variables were also performed. Means 6 SEM were considered different when P , 0.05 unless otherwise stated.

Control fed 3.62 1.60 5.82 174.60 91.51 70.84 1085.69 568.04 1607.87 157.73 285.74 625.43 94.94

6 6 6 6 6 6 6 6 6 6 6 6 6

Nutrient restricted

0.37 0.11 0.70 9.69 4.77 3.75 67.17 48.18 106.03 10.43 23.04 74.13 8.11

2.87 1.40 4.54 168.32 90.64 62.86 778.77 442.52 1220.82 123.38 259.88 659.03 108.17

6 6 6 6 6 6 6 6 6 6 6 6 6

0.30 0.15 0.34 10.02 8.00 4.85 70.21a 46.37 150.52 14.02 36.44 78.33 20.77

Significantly different compared with control-fed ewes (P # 0.05).

RESULTS

On Day 28, prior to the onset of nutrient restriction, the weight of the ewes did not differ between the control-fed (n 5 7) and nutrient-restricted groups (n 5 6; 93.90 6 4.66 vs. 91.56 6 5.35 kg, respectively). At the end of the study, the live weight of control-fed ewes was greater (P , 0.05) than that of nutrient-restricted ewes (102.08 6 4.98 vs. 85.83 6 5.01 kg; respectively), as a result of a 7.51 6 1.29% body weight gain in the control-fed ewes and a 7.38 6 0.87% body weight loss in nutrient-restricted ewes (Fig. 2). This difference in live weight between the groups is also reflected by a loss in empty body weight (weight after removal of the viscera; 65.97 6 3.24 vs. 57.36 6 4.39 kg) for control-fed and nutrient-restricted ewes, respectively. Absolute maternal organ weights were similar between control fed and nutrient restricted ewes (Table 1) with the exception of the liver, which was reduced (P , 0.05) in nutrient-restricted ewes. If individual organ weights are divided by the weight of the ewe, to correct for body weight, liver weights were also similar between the control-fed and nutrient-restricted ewes. The number of fetuses gestated by each ewe on Day 45, as determined by ultrasonography, was identical to the number present on Day 78, demonstrating no fetal loss during the subsequent course of nutrient restriction. Additionally, the lack of fetal loss was confirmed by counting the number of corpora lutea on the ovaries of each ewe at tissue collection. The control-fed group had three singleton and four twin pregnancies, while the nutrient-restricted group had four singleton and two twin pregnancies. Concentrations of E2-17B and P4 in systemic circulation of nutrient-restricted and control-fed ewes were similar immediately prior to killing (Table 2). In contrast, nutrientrestricted ewes had decreased (P , 0.05) plasma glucose concentrations and an increased (P , 0.05) plasma T4/T3 TABLE 2. Maternal glucose and hormone concentrations at slaughter from Day 78 of gestation in control-fed (n 5 7) and nutrient-restricted (n 5 6) ewes. Maternal values Estradiol-17b, pg/ml Progesterone, ng/ml Glucose, mg/dl T3, nM T4, nM T4/T3 a

Control fed 24.24 5.75 49.54 1.09 45.51 42.29

6 6 6 6 6 6

4.63 0.42 2.33 0.13 5.27 2.18

Nutrient restricted 37.67 6.96 43.38 0.92 49.03 54.00

6 6 6 6 6 6

9.47 0.99 2.17a 0.05 2.17 2.63a

Significantly different compared with control-fed ewes (P , 0.05).

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TABLE 3. Fetal glucose and hormone concentrations from fetal cord blood and amnionic fluid on Day 78 of gestation from control-fed and nutrientrestricted ewes.a Control fed

Nutrient restricted

Fetal values

Singles (n 5 3)

Twins (n 5 8)

Singles (n 5 4)

Twins (n 5 4)

Glucose, mg/dl T3, nM T4, nM Cord blood VEGF, ng/ml Estradiol 17b, pg/ml

18.06 6 0.75 ND 21.23 6 1.54 0.68 6 0.15 80.84 6 29.07

16.50 6 1.22 ND 23.41 6 2.31 0.61 6 0.07 60.94 6 7.20

13.38 6 ND 23.18 6 1.22 0.66 6 0.04 81.54 6 47.26

13.33 6 0.69b ND 13.93 6 1.28c 0.56 6 0.07 63.69 6 23.88

1.12b

a

ND, nondetectable levels. Different from control fed (P , 0.05). c Different from control-fed singles, control-fed twins, and nutrient-restricted singles (P , 0.05). b

Total numbers of placentomes in gravid uteri of controlfed ewes were similar to numbers found in nutrient restricted ewes (88 6 3 vs. 80 6 4, respectively). Further, numbers of placentomes associated with singleton fetuses (84 6 5) were double (P , 0.01) the numbers associated with a twin fetus (42 6 2) across both treatment groups. While similar numbers of placentomes were associated with twin fetuses of control-fed and nutrient-restricted ewes, numbers of placentomes were greater (P , 0.05) for singleton fetuses from control-fed ewes when compared to the number in nutrient-restricted singleton fetuses from nutrient-restricted ewes (92 6 4 vs. 78 6 6, respectively). There was no difference in the range or distribution of placentomal weight between conceptuses of control-fed or nutrient-restricted ewes (Fig. 3). Similar to placentomal weight, there was no difference in the range or distribution of placentomal surface area between conceptuses of the control-fed and nutrient-restricted groups (Fig. 4). Further, there was no effect of twin versus singleton pregnancies on the range or distribution of placentomal weight or surface area. There were no effects of treatment or fetal number on the placentomal types observed, with virtually all placentomes being classified as type A (i.e., caruncular tissue completely surrounding the cotyledonary tissue) for all animals. In contrast, a significant effect (P , 0.05) of twin versus single pregnancies was observed in the surface area of individual placentomes (Fig. 5). Individual placentomes associated with a singleton conceptus had reduced (P , 0.05) surface areas than those associated with a twin conceptus (i.e., a greater percentage of smaller placentomes [,10 cm2] were associated with the singleton fetuses vs. twin fetuses). In agreement with the data presented here, there were no differences (P . 0.05) in total placentome weight and surface area or total caruncular and cotyledonary weights

ratio when compared to control-fed ewes (Table 2). Similarly, glucose concentrations in fetal blood were also decreased (P , 0.05) in fetuses from nutrient-restricted versus control-fed ewes (Table 3). While T3 concentrations were undetectable in fetal blood, T4 concentrations were decreased (P , 0.05) in twin fetuses from nutrient-restricted ewes when compared with singleton and twin fetuses from control-fed ewes and singleton fetuses from nutrient-restricted ewes (Table 3). Vascular endothelial growth factor and E2-17B concentrations in fetal blood were similar across singleton and twin pregnancies of both treatment groups, averaging 0.63 ng/ml and 71.75 pg/ml, respectively. Fetuses from control-fed ewes were markedly (P , 0.01) heavier and larger (. crown rump length, CRL) than fetuses from nutrient-restricted ewes (Table 4). However, there was no difference in abdominal circumference of the fetuses from control-fed or nutrient-restricted ewes. There was no difference (P . 0.10) in weight or CRL between singleton and twin fetuses gestated by control-fed ewes (325.97 6 34.99 g and 23.00 6 1.26 cm vs. 326.51 6 25.57 g and 23.75 6 0.54 cm, respectively). In contrast, singleton fetuses were heavier (P , 0.05) and tended to be larger (P , 0.10) than twin fetuses in the nutrient-restricted group (240.75 6 2.90 g and 22.38 6 1.03 cm vs. 202.60 6 6.37g and 20.75 6 0.48 cm, respectively). Fetal liver, lungs, and kidneys from nutrient-restricted ewes were reduced (P , 0.05) in weight when compared to those of control-fed ewes (Table 4). When corrected for fetal weight, however, all organ weights evaluated were similar between fetuses gestated by nutrient-restricted and control-fed ewes with the exception of the liver and the right and left ventricles of the heart, which were greater (P , 0.05) per unit fetal weight in the nutrient-restricted ewes.

TABLE 4. Fetal organ weights from conceptuses on Day 78 of gestation from control-fed and nutrient-restricted ewes.a Measurements Control (n 5 11) Fetal weight, g CRL, cm Abdom circ, cm Liver, g Pancreas, g Lung, g Kidney, g Adrenal, g Left ventricle, g Right ventricle, g Septum, g a b

326.36 23.55 14.15 19.50 0.34 16.17 3.55 0.15 1.01 0.66 0.51

6 6 6 6 6 6 6 6 6 6 6

19.99 0.50 0.34 0.97 0.04 1.06 0.17 0.03 0.15 0.08 0.07

Abdom circ, abdominal circumference. Means 6 SEM differ within a measure (P , 0.05).

Organ weight/fetal weight Restricted (n 5 8)

Control (n 5 11)

Restricted (n 5 8)

6 6 6 6 6 6 6 6 6 6 6

— — — 6.02 6 0.13 0.11 6 0.01 4.98 6 0.18 1.10 6 0.05 0.05 6 0.01 0.30 6 0.03 0.20 6 0.02 0.15 6 0.01

— — — 6.71 6 0.37b 0.14 6 0.04 5.13 6 0.33 1.10 6 0.09 0.04 6 0.01 0.41 6 0.03b 0.25 6 0.02b 0.17 6 0.02

221.68 21.56 13.16 14.97 0.31 11.41 2.65 0.09 0.91 0.57 0.37

7.90b 0.61b 0.43 1.19b 0.08 0.90b 0.26b 0.02 0.08 0.07 0.05

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FIG. 3. Range and distribution (mean 6 SEM) of placentome weights from control-fed (solid symbols) and nutrient-restricted (open symbols) ewes on Day 78 of gestation.

between conceptuses from control-fed and nutrient-restricted ewes (Table 5). As would be expected from the greater numbers of placentomes present, singleton fetuses, regardless of treatment, had markedly (P , 0.01) heavier total placentome weight as well as caruncular and cotyledonary weights than twin fetuses. Table 5 depicts the ratios of fetal weight to cotyledonary, caruncular, and total placentomal weights. Conceptuses from nutrient-restricted ewes exhibited reduced ratios of fetal weight/cotyledonary weight, fetal weight/caruncular weight, and fetal weight/total placentome weight when compared to conceptuses from control-fed ewes. Although nutrient restriction did not affect the size or morphologic type of placentomes on Day 78, placentomal vascularity was impacted. There was a 3-fold increase (P , 0.05) in the number of caruncular blood vessels per unit area tissue of twin conceptuses from nutrient-restricted ewes when compared to singleton and twin conceptuses

FIG. 5. Range and distribution (mean 6 SEM) of placentome surface area from singleton (solid bars) and twin (open bars) conceptuses on Day 78 of gestation. *, different from singles (P , 0.05).

recovered from control-fed ewes or singleton conceptuses recovered from nutrient-restricted ewes (Table 6). The number of cotyledonary blood vessels per unit area tissue did not differ between groups. There was no difference in the area of blood vessels per unit tissue area in either the caruncular or the cotyledonary tissue in any of the fetal groups. Further, while caruncular blood vessel diameter was larger (P , 0.05) than cotyledonary blood vessel diameter, there was no treatment effect in blood vessel diameter of either the caruncular blood vessels (266.40 6 31.50 mm) or the cotyledonary blood vessels (70.37 6 5.40 mm). DISCUSSION

To our knowledge, these data are the first to demonstrate a significant reduction in fetal weight immediately following a prolonged bout of nutrient restriction from early to mid-gestation. In contrast to these data, Clarke et al. [18] provided 60% of nutrient requirements during an equivalent period of gestation but found no differences in fetal size or weight on Day 78. The marked reduction in fetal weight observed in the present study may have resulted from the more severe nutrient restriction utilized in our model. This is consistent with the observed decrease in maternal body weight as well as the reduced glucose concentrations and the significantly increased T4/T3 ratio in maternal blood of nutrient-restricted ewes when compared to concentrations in control-fed ewes. In contrast to these data, while both T4 and T3 concentrations in maternal blood were reduced TABLE 5. Conceptus measurements on Day 78 of gestation from controlfed (n 5 7) and nutrient-restricted (n 5 6) ewes. Control fed Total placentome weight, g Total placentome surface area, cm2 Total cotyledonary tissue weight, g Total caruncular tissue weight, g Fetal weight/cotyledonary weight Fetal weight/caruncular weight Fetal weight/total placentome weight

FIG. 4. Range and distribution (mean 6 SEM) of placentome surface area from control-fed (solid symbols) and nutrient-restricted (open symbols) ewes on Day 78 of gestation.

1003 879 667 176 0.81 3.23 0.53

6 6 6 6 6 6 6

139 97 100 14 0.08 0.33 0.05

Nutrient restricted 815 714 579 138 0.54 2.21 0.37

6 6 6 6 6 6 6

111 77 87 15 0.08a 0.35b 0.05a

Control-fed significantly different from nutrient-restricted ewes (P , 0.05). b Control-fed ewes differ from nutrient-restricted ewes (P 5 0.065). a

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TABLE 6. Vascular measurements of placentomes from single and twin fetuses from control-fed and nutrient-restricted ewes on Day 78 of gestation. Control ewes Maternal measurements Vessel numbers per unit area Vascular density (vessel area/tissue area) Fetal Measurements Vessel numbers per unit area Vascular density (vessel area/tissue area) a

Restricted ewes

Singles (n 5 3)

Twins (n 5 8)

Singles (n 5 4)

Twins (n 5 4)

8.21 6 3.50 3 1026 0.25 6 0.08

6.67 6 1.29 3 1026 0.23 6 0.03

5.28 6 8.30 3 1026 0.23 6 0.02

23.32 6 10.26 3 1026 a 0.22 6 0.05

7.35 6 1.84 3 1026 0.04 6 0.02

7.37 6 1.26 3 1026 0.03 6 0.01

5.94 6 0.73 3 1026 0.02 6 0.004

10.35 6 2.88 3 1026 0.04 6 0.01

Twin fetuses from nutrient restricted ewes differ from other fetal groups (P , 0.05).

in the studies of Clarke et al. [18] and Heasman et al. [19], no differences in the plasma T4/T3 ratio were apparent between nutrient-restricted and control-fed ewes. As most of the circulating T3 is derived from peripheral deiodination of T4, a greater inhibition of 59-deiodinase or an increase in monodeiodination involved in reverse(r)T3 production must be occurring in peripheral tissues of our nutrient-restricted ewes [29–31]. Concentrations of T4 were reduced in the blood of twin fetuses gestated by nutrient-restricted ewes when compared to fetuses from control-fed ewes or singleton fetuses from nutrient-restricted ewes, suggesting a further nutrient associated reduction in fetal metabolic rate in these twins. Most of the T4 in fetal mammals is deiodinated to the inactive metabolite, rT3, via an iodothyronine 5-monodeiodinase in fetal tissue [32, 33]. In the fetal sheep, the ability to convert T4 to active T3 does not occur until the third trimester of gestation [33], which explains our inability to detect T3 in fetal blood on Day 78 of gestation. While Clarke et al. [18] and Heasman et al. [19] progressively increased the amount of feed given to each ewe with advancing gestation to comply with the Agricultural and Food Research Council [34] guidelines, we adjusted feed intake up (control fed) or down (nutrient restricted) based only on gains or losses in ewe body weight. Orskov and Ryle [35] reported that the ruminant becomes more efficient in utilizing nutrients when energy requirements are not met (e.g., during fasting). Thus, in contrast to Clarke et al. [18], who reported no differences in ewe body weight regardless of dietary regimen, nutrient-restricted ewes in our study lost a significant amount of weight, while controlfed ewes gained an equivalent amount of weight during the treatment period in accordance with the advancement of normal pregnancy in sheep. As the average control-fed ewe in this study gained ;7.5% of her initial body weight by Day 78 of gestation, one could consider that our nutrientrestricted ewes actually lost ;15% of predicted weight (;7.5% predicted weight gain plus 7.4% actual weight loss). The reduced body weight in the nutrient-restricted ewes is not due strictly to gastrointestinal tract fill, as the empty body weight at the end of the study was also reduced, indicating a loss of fat and/or muscle in the nutrientrestricted ewes compared to the control-fed ewes. Fetuses from nutrient-restricted ewes were markedly lighter than those from control-fed ewes but exhibited individual organs that were not uniformly reduced in weight. Increased liver weight per unit fetal weight in fetuses from nutrient-restricted ewes compared to control-fed ewes may be explained by an increase in liver metabolic activity imperative for development. It has been reported that glucose requirements of the growing ovine fetus are met largely via placental uptake of maternal glucose [36] and that maternal and fetal blood glucose concentrations are highly correlated [37]. Lemons et al. [38] reported that when ewes are fasted

at 120 days of gestation, the gluconeogenic enzymes (e.g., glucose-6-phosphatase, glutamate pyruvate transaminase, phosphoenolpyruvate carboxykinase) are increased in fetal liver. As ruminants depend primarily on gluconeogenesis rather than intestinal glucose absorption to meet their glucose requirement [39], it was suggested that even the fetal liver has the potential to perform this metabolic function in utero when glucose requirements are not met. Further investigation into the gluconeogenic properties of the fetal liver is warranted. Of interest is the observation that the right and left ventricles of the heart were larger per unit body weight in fetuses from nutrient-restricted than control-fed ewes, thereby demonstrating a bilateral ventricular hypertrophy. This condition is suggestive of increased ventricular afterload (i.e., the force that a ventricle must overcome while it contracts during ejection), seen in human fetuses using high-resolution echocardiography [40]. The components that contribute to increased ventricular afterload are aortic or pulmonary artery impedance, peripheral vascular resistance, and mass and viscosity of blood. The observation of bilateral ventricular hypertrophy is exciting, as Samson et al. [41] reported that no animal model has been developed that reproduces the observations in the human fetus associated with increased left-ventricular afterload. Previous models using the fetal lamb have invariably resulted in leftventricular hypertrophy without right-heart enlargement [42, 43]. Thus, this model of early fetal nutrient deprivation in the sheep may also serve as a relevant model to study the etiology and consequences of human fetal ventricular hypertrophy. We hypothesize that the bilateral increase in fetal ventricular weights from the nutrient-restricted ewes may be due to an increase in placental vascular resistance. Rigano et al. [44] demonstrated that umbilical vein blood flow is reduced in intrauterine growth-restricted (IUGR) fetuses as a result of reduced umbilical vein blood flow velocity. Further, reduced umbilical blood flow, increased umbilical arterial pulsatility index, and increased placental vascular resistance have been reported for both human and sheep IUGR pregnancies [45–48]. The reduced ratios of fetal weight to cotyledonary, caruncular, and total placentome weight on Day 78 of gestation indicate that there is a reduced placentomal function of the conceptuses from nutrient-restricted ewes, possibly due to a decrease in the amount of nutrients delivered to the developing fetus. Although no diet-associated differences in placental weight or placentome numbers, sizes, or morphologies were observed in the present study, we did observe that twin conceptuses had reduced numbers of placentomes, with a greater percentage of those placentomes having a larger surface area than those associated with singleton fetuses. Further, marked increases were observed in the caruncular vascular density of placentomes from twin conceptuses gestated by


nutrient-restricted ewes. The number of caruncular, but not cotyledonary, blood vessels per unit area in nutrient-restricted ewes carrying twins was ;3-fold greater than that in control-fed ewes carrying singles or twins or nutrientrestricted ewes carrying twins. Morris et al. [49] demonstrated a 25% reduction in uterine blood flow and a 20% decrease in placental blood flow of ewes fasted for 5 days prior to evaluation on 100 days of gestation. The decrease in nutrient availability, as well as the stress of carrying twins, may have increased caruncular vascularity in order to compensate for decreased nutrient delivery. Despite the increase in caruncular vascularity, there was no corresponding increase in the numbers of cotyledonary blood vessels in placentomes of restricted twins, potentially negating the impact of increases in caruncular vascular numbers on Day 78 of gestation. However, since cotyledonary vascularity increases markedly after Day 80 in the ewe [9], overall vascularity of the placentome may be greater in the nutrient-restricted ewes compared to control-fed ewes by term. Vascular endothelial growth factor is a potent angiogenic factor at the fetal:maternal interface in both cotyledonary and caruncular tissue [50], and its expression is known to be increased by estrogen [16] and hypoxia [17]. In the pig conceptus, both VEGF mRNA [51] and protein expression (S.P. Ford, unpublished observations) are highly correlated with placental vascularity late in gestation (Days 70–110 of gestation). Both fetal demand for oxygen and placental secretion of estrogen increase dramatically in late gestation in both the pig and the sheep. In this regard, we have demonstrated no treatment differences in P4 or E2-17B concentrations in either fetal or maternal blood, suggesting that there is no interruption in normal steroidogenesis occurring between our control-fed and nutrient-restricted ewes. Further, we observed no differences in VEGF concentrations in fetal cord blood from nutrient-restricted and control-fed ewes. This observation is not surprising, as Carnegie and Robertson [52] show a dramatic increase in fetal plasma estrogen only after Day 80 of gestation in the ewe. This dramatic increase in placental estrogen production after Day 80 of gestation is temporally associated with the marked increases in cotyledonary VEGF mRNA expression [15], cotyledonary vascular density [9], and uterine blood flow [53]. It is possible that the increased caruncular vascularity of twin conceptuses from nutrient-restricted ewes in this study might facilitate an augmented nutrient delivery to these undersized fetuses during a realimentation after Day 80, when cotyledonary vessels begin to proliferate. A markedly increased nutrient delivery, to the previously undernourished twin fetuses, could then result in compensatory fetal growth throughout the second half of gestation, resulting in average to above-average birth weights. Although the offspring from nutrient-restricted ewes may be born at similar or greater weights than the offspring from control-fed ewes, it is quite possible that the cardiac ventricular hypertrophy and increased liver weight we observed in fetuses from nutrient-restricted ewes on Day 78 of gestation may lead to hypertension, coronary heart disease, stroke, and/or metabolic disruptions either in neonatal or in adult life. However, further studies will be required to evaluate this hypothesis. In summary, a constant reduction in energy intake during the critical period of organogenesis and placental development in the pregnant ewe results in markedly smaller fetuses with an accelerated growth of the left and right ventricles of the heart as well as the fetal liver. Further, an enhanced vascular density was observed in the caruncular

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