The effect of maternal undernutrition on the placental growth trajectory and the uterine insulin-like growth factor axis in the pregnant ewe.

The placenta is a highly efficient multifunctional organ, mediating the exchange of nutrients, gases and waste products between the dam and fetus. This study investigated the effects of chronic maternal undernutrition (70% of estimated requirement) on the placental growth trajectory in the ewe on days 45, 90 and 135 of gestation. The insulin-like growth factor (IGF) system was investigated using in situ hybridisation analysis to determine if nutritionally mediated alterations in placental growth were regulated through modifications in placental IGF expression. Placental weight increased between days 45 and 90 (P<0.01), accompanied by a reduction in maternal placentome IGF binding protein (IGFBP)-3, -5 and -6 expression (P<0.05), although IGF-II mRNA levels in maternal villi remained unchanged. Placentome number was unaffected by diet or gestational age. Placental weight remained constant between days 90 and 135 in ewes on 100% maintenance rations but decreased over this period (P<0.05) in ewes on the 70% rations. Gross morphology also altered, so the underfed ewes had more type C and type D placentomes and fewer type B placentomes than their well-fed counterparts on day 135 (P<0.05). These changes were accompanied by higher IGFBP-6 mRNA expression in the maternal placental villi in undernourished ewes (P<0.05). The change in shape from a type A to a type C placentome was accompanied by flattening of the placentome and a reduction in the ratio of the area of unattached fetal allantochorion to interdigitated maternal and fetal villi. Within the intercotyledonary endometrium, expression of IGFBPs-3 and -5 mRNA in the glandular epithelium increased between days 45 and 90, showing an opposite trend with time to that found in the adjacent placentomes. This indicates tissue-specific control of IGFBP expression. In conclusion, this study has shown clear time-related changes in the uterine IGFBP system during pregnancy, which accompany changes in placental growth. Altered IGFBP expression may play a role in determining placental size in relation to nutritional status, but is unlikely to be the only mediator.

Alterations in placental 11 beta-hydroxysteroid dehydrogenase (11 betaHSD) activities and fetal cortisol:cortisone ratios induced by nutritional restriction prior to conception and at defined stages of gestation in ewes.

In the placenta, cortisol is inactivated by NADP(+)- and NAD(+)-dependent isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD). Decreased placental 11betaHSD activities have been implicated in intrauterine growth restriction (IUGR) and fetal programming of adult diseases. The objective of this study was to investigate whether placental 11betaHSD activities and fetal plasma cortisol:cortisone ratios could be affected by nutritional restriction of ewes (70% maintenance diet) throughout gestation, for specific stages of gestation, or prior to mating. Chronic nutritional restriction from day 26 of gestation onwards decreased NAD(+)-dependent 11betaHSD activities by 52 +/- 4% and 45 +/- 6% on days 90 and 135 of gestation respectively. Although the decreases in enzyme activities were associated with fetal IUGR, the cortisol:cortisone ratio in fetal plasma was unaffected by chronic nutritional restriction throughout pregnancy. Nutritional restriction confined to early (days 26-45), mid- (days 46-90) and late gestation (days 91-135), or the 30 days prior to mating, had no significant effect on NAD(+)-dependent, placental 11betaHSD activities, nor was there evidence of IUGR. However, nutritional restriction at each stage of pregnancy and prior to mating was associated with significant decreases in the fetal plasma cortisol:cortisone ratio (3.2 +/- 0.7 in control fetuses; 1.0 to 1.6 in fetuses carried by nutritionally restricted ewes). We conclude that nutritional restriction of pregnant ewes for more than 45 consecutive days can significantly decrease NAD(+)-dependent placental 11betaHSD activities in association with IUGR. While the cortisol:cortisone ratio in fetal plasma is sensitive to relatively acute restriction of nutrient intake, even prior to mating, this ratio does not reflect direct ex vivo measurements of placental 11betaHSD activities.

Effect of maternal body condition on placental and fetal growth and the insulin-like growth factor axis in Dorset ewes.

This study investigated the effects of maternal body condition on fetal growth. Fetal and placental parameters from Dorset ewes of body condition score 2.0 (lean, n = 5), 3.5 (moderate, n = 7) and 5.0 (fat, n = 4) at mating were studied on day 65 of gestation. The fetal weight and fetal weight:crown-rump length ratio were greater in fat ewes than in ewes of moderate condition. The raised total and mean placentome weight in fat ewes compared with ewes of moderate condition may have contributed to their increased fetal growth. However, the fetal crown-rump length was not affected. With in situ hybridization, insulin-like growth factor II (IGF-II) mRNA and insulin-like growth factor binding protein 2 (IGFBP-2), -3 and -6 were all detected in the placentome capsule; IGF-II mRNA was also found in the mesoderm of the fetal villi and IGFBP-3 and IGFBP-6 were present in the caruncular stroma of the maternal villi. Ewes of moderate condition, which had the smallest placentae, had the greatest placental expression of IGF-II, IGFBP-2 and IGFBP-3. In the intercotyledonary endometrium, IGFBP-3, IGFBP-5 and uterine milk protein (UTMP) mRNA were all expressed in the glandular epithelium. IGFBP-3 and IGFBP-5 absorbance values were lowest in the lean ewes, whereas UTMP values were highest. Maternal insulin concentrations were greater in fat ewes, whereas plasma glucose and IGF-I concentrations in the fetal compartment were lowest in fat ewes. Therefore, in obese ewes, fetal and placental growth is increased in mid-gestation in association with higher maternal insulin concentrations and lower expression of IGFBPs in the maternal placentomes. Placental and fetal development in lean ewes may be promoted by reduced IGFBP expression in the placentomes and enhanced UTMP production by the endometrial glands. The ewes of moderate condition had the smallest fetuses and placentae coupled with the highest placental expression of IGF-II and IGFBPs.

The effects of maternal nutrition and body condition on placental and foetal growth in the ewe.

This study investigated the effects of maternal body condition and nutrition on placental and foetal growth in mid-gestation. Welsh Mountain ewes (n=24) of body condition 3.5 (high, H) and 2.0 (low, L) at mating, were fed either 100 per cent or 70 per cent of their daily maintenance requirements from day 22 of gestation, yielding four groups: H100 (n=5), H70 (n=6), L100 (n=7) and L70 (n=6). On day 65, placental and foetal parameters were measured. Whilst the placentome number tended to be lower in L than H ewes, the mean placentome weight was significantly greater in L100 than H100 animals. Nutritionally related changes in IGFBP expression within the placentome and intercotyledonary endometrium may explain these findings, with IGFBP-3 expression in the luminal epithelium and caruncular stroma of the placentome villi being inversely correlated to placentome number and the total placentome weight respectively. The foetal CRL was shorter and the ponderal index greater in L than H ewes. The foetal CRL was positively correlated to maternal IGF-I concentrations and the placentome number, although the foetal weight remained unaltered by treatment. This study therefore demonstrates that body condition and ration can alter foetal and placental growth, perhaps by modifying systemic parameters and uterine IGF expression.

Regulation of insulin-like growth factor binding protein-6 expression in the reproductive tract throughout the estrous cycle and during the development of the placenta in the ewe.

Insulin-like growth factors (IGF-I and IGF-II) are essential for normal uterine development and have been particularly implicated in fetal and placental growth. A family of six IGF binding proteins enhance or attenuate IGF-stimulated cell proliferation. In this study we have used in situ hybridization to map the distribution of IGFBP-6, one of the lesser known of the IGFBPs, in sections of the uterus collected from cyclic, anestrous, and ovariectomized nonpregnant ewes and from the uterus and placenta of early pregnant (13-55 days) and unilaterally pregnant ewes. In nonpregnant ewes IGFBP-6 mRNA (measured as arbitrary optical density units from autoradiographs) was abundant in the periepithelium and caruncles, with lower levels in the endometrial stroma and myometrium. In most regions IGFBP-6 mRNA showed cyclic variations with concentrations maximal around ovulation and the early luteal phase. In addition, 16 out of 25 ewes expressed IGFBP-6 mRNA in their endometrial glands between estrus and Day 2. Measurements of IGFBP-6 mRNA were high in anestrous ewes (equivalent values to ovulation) but low in ovariectomized ewes (equivalent values to mid to late luteal phase). In pregnant ewes IGFBP-6 mRNA was found in similar regions to those recorded during the cycle. In the periepithelium and caruncular stroma IGFBP-6 mRNA levels were higher during early pregnancy than in the midluteal phase. In the unilateral pregnant ewes there was no difference in IGFBP-6 mRNA measured between pregnant and nonpregnant horns. In conclusion, IGFBP-6 mRNA is differentially regulated during the estrous cycle and pregnancy and may be functionally important in modulating IGF activity in the uterus and placenta by virtue of its strong affinity and ability to regulate IGF-II mediated actions.

The effect of maternal undernutrition on ovine fetal growth.

Modifications in maternal nutrition during pregnancy can significantly disrupt fetal growth and subsequent post-natal health and survival. This study investigated the effects of undernutrition on fetal growth and the potential mechanisms involved. Tissue from pregnant ewes (n=27) was investigated on days 45, 90 and 135 of gestation (term = approximately 150 days). The thoracic girth (P<0.05) was greater in fetuses from nutrient restricted ewes on day 45 and there was also a trend towards an increased gut weight (P<0.08). By day 90, the fetal brain and thymus weight were lighter in underfed than in well-fed animals whilst the weight of the fetal ovaries was heavier (P<0.05). On day 135 the fetal heart, pancreas, thymus, gut and kidney weights were lighter in undernourished ewes (P<0.05). When expressed as a percentage of fetal body weight, significance was retained in the heart, pancreas and thymus (P<0.05). Bone growth was also affected. At day 90 the fetal femur and metatarsal were longer in underfed mothers (P<0.05). In contrast, the fetal humerus and scapula were shorter in underfed than in well-fed animals on day 135 (P<0.05) when the weight of the semitendinosus muscle (P<0.05) was also reduced. The fall in fetal glucose (P<0.1), insulin (P<0.01) and IGF-I (P<0.01) levels in underfed ewes on day 135 may have compromised fetal growth. Fetal plasma IGF binding protein-2 also increased between days 90 and 135 in underfed ewes (P<0.03), whilst levels were unaltered in well-fed animals. Although maternal and fetal plasma IGF-I levels increased with gestation (P<0.01) and the placentome morphology altered in all ewes (P<0.05), the fall in placental mass (P<0.05), amniotic and allantoic glucose concentrations (P<0.05) and maternal plasma glucose and insulin levels (P<0.05) in underfed ewes in late gestation may have compromised fetal substrate delivery. These perturbations in fetal development may have significant implications on adult health and carcass conformation, raising important health and economic issues in medical and agricultural sectors.

IGF-I stimulation of luteinizing hormone secretion, IGF-binding proteins (IGFBPs) and expression of mRNAs for IGFs, IGF receptors and IGFBPs in the ovine pituitary gland.

Circulating concentrations of insulin-like growth factor-I (IGF-I) are reduced in juvenile sheep during nutritional growth restriction and the associated delay in puberty. Since exogenous IGF-I has been shown to stimulate luteinizing hormone (LH) secretion, it is postulated that endogenous IGF-I may act as a stimulatory metabolic signal to the pubertal ovine hypothalamo-pituitary axis, yet its site of action is unknown. Using coronal hypothalamic and pituitary sections from pubertal ewe lambs, in vitro autoradiography was used to localise 125I-labelled IGF-I binding, and gene expression for components of the IGF system was localised by in situ hybridisation using oligonucleotide probes. High concentrations of 125I-IGF-I binding were seen in the pars tuberalis (PT) and pars distalis (PD) of the pituitary, and relatively little in the hypothalamus; binding in the PT but not the PD was displaced by excess unlabelled IGF-I. Large amounts of mRNA were detected for the type-1 receptor (IGF-1R) and for IGF-binding protein (IGFBP)-5, localised to the PT and PD, and less intense specific hybridisation signals were obtained with mRNAs for IGF-II, type-2 receptor (IGF-2R) and IGFBP-3. There was some evidence for specific hybridisation to IGFBP-4 mRNA in the PT. IGF-I, IGFBP-1 and IGFBP-2 mRNAs were not detected in PT and PD. None of the genes were expressed in hypothalamic tissue. Western-ligand binding on PD extracts from male castrates revealed by their molecular weights the likely presence of IGFBPs-2, -3, and -5. Finally, cultured PD cells from abattoir-killed sheep were challenged with IGF-I (0.1, 1, 10 or 30 nM) or luteinizing hormone-releasing hormone (LHRH, 10 nM) alone, or both together. Basal LH output was stimulated by 10 nM IGF-I (120+/-11.2%, P>0.05), 30 nM IGF-I (148+/-12.8%, P<0.01), and LHRH alone (200+/-16.1%, P<0.001); there was no additive or subtractive effect of LHRH and IGF-I given together. Thus, an intrapituitary IGF system exists in sheep and the present results are consistent with an endocrine role for IGF-I in nutritional modulation of LH secretion at the level of the pituitary gland.

Effect of a high maternal dietary intake during mid-gestation on components of the utero-placental insulin-like growth factor (IGF) system in adolescent sheep with retarded placental development.

The aim of the present study was to investigate the effects of administering a high plane diet during early to mid-gestation on the uterine and placental insulin-like growth factor (IGF) system and on systemic IGF-I concentrations in pregnant adolescent ewes with restricted placental growth. Embryos recovered from superovulated ewes inseminated by a single sire were transferred in singleton to the uterus of adolescent recipients. After transfer ewes were offered a high (H) or moderate (M) amount of a complete diet calculated to promote rapid or normal maternal growth rates, respectively. Five ewes from each group were switched from either M to H or H to M diets at day 52 of gestation. Maternal and fetal blood samples and placental tissues were collected from all animals at day 104. Ewes on the high plane diet from mid-gestation (HH, MH groups) had restricted placental mass (P < 0.01) and tended to have smaller fetuses. This was associated with increased maternal plasma IGF-I concentrations (P < 0.001). The pattern of expression of components of the IGF system in the uterus and placenta was studied by in situ hybridization. IGF-I mRNA concentrations were below the limit of detection. IGF-II mRNA expression was high in the fetal mesoderm and present in maternal stroma, but was not influenced by nutritional treatment. In contrast, IGF binding protein 1 (IGFBP-1) mRNA expression was higher (P < 0.05) and IGFBP-3 mRNA expression was lower (P < 0.05) in the endometrial glands of ewes in HH and MH groups. In the fetal trophoblast, IGFBP-3 mRNA expression was higher in the MH group. Type 1 IGF receptor expression was increased (P < 0. 01) in the luminal epithelium of the HM group and IGFBP-2 mRNA expression was highest in the placentome capsule of ewes in the HH group. Together, these results indicate that reprogramming of the uterine and placental IGF axis by maternal nutrition could contribute to placental growth retardation in growing adolescent sheep.

The expression of the IGF system in the bovine uterus throughout the oestrous cycle and early pregnancy.

The IGF system is expressed in the uterus during the oestrous cycle and early pregnancy and is likely to play an important role in regulating the development of the embryo and uterus. The IGF peptides (IGF-I and -II) mediate their effects through the type 1 IGF receptor (IGF-1R), while the IGF-binding proteins (IGFBP-1 to -6) modulate their interaction with the receptor. In this study, the expression of the IGF system in the bovine uterus was determined throughout the oestrous cycle and on day 16 of pregnancy. Endometrial biopsy samples were collected from four cows over three cycles such that there were samples for every 2 days from day 0 (oestrus) to day 14 and then every day until day 21. To assess the effect of pregnancy, uterine horn cross-sections were collected on day 16 from 15 pregnant (PREG), five inseminated non-pregnant (INP) and nine uninseminated cyclic controls (CONT). The expression of mRNA for the IGFs, IGF-1R and IGFBP-1 to -5 was determined by in situ hybridisation and the results were quantified by measuring the optical density units from autoradiographs. The main region of IGF-I mRNA expression was the sub-epithelial stroma underlying the luminal epithelium. The expression of IGF-I mRNA was highest at oestrus and lowest during the early and late luteal phases. On day 16, IGF-I mRNA levels were low in all groups, with pregnancy having no effect on the IGF-I mRNA concentrations. The strongest expression of IGF-II mRNA was in the caruncular stroma, with pregnancy having no significant effect in this region. IGF-1R mRNA was also present in the caruncles and was strongly expressed in all epithelial cells both throughout the oestrous cycle and during early pregnancy. The expression of IGFBP-1 mRNA was confined to the luminal epithelium, with the strongest expression seen on day 14 of the cycle. On day 16 the expression of IGFBP-1 mRNA was higher in the PREG group compared with the CONT group. The expression of IGFBP-2 mRNA was localised to the sub-epithelial stroma with more INP than PREG cows showing detectable levels of IGFBP-2. The strongest expression of IGFBP-3 mRNA was in the caruncular stroma; expression in the endometrial stroma was similarly decreased during early pregnancy. IGFBP-5 mRNA was mainly expressed in the inner ring of myometrium and was not affected by pregnancy on day 16. In conclusion, these results show that many components of the uterine IGF system are differentially regulated during the oestrous cycle and early pregnancy and suggest that modulation of the IGF system may influence uterine activity during this period.

Regulation and localization of insulin-like growth factor binding protein-5 gene expression in the uterus and placenta of the cyclic and early pregnant ewe.

The insulin-like growth factor (IGF) system plays an important role in the regulation of uterine function and placental growth. However, there is little information regarding the localization and regulation of IGF binding protein-5 (IGFBP-5) in the reproductive tract. The distribution of this IGFBP was therefore investigated using in situ hybridization in sections of utero-placental tissue obtained throughout the estrous cycle, up to Day 55 of gestation, and on Days 16-17 from both horns of ewes with unilateral pregnancies that followed uterine transection. In nonpregnant ewes, IGFBP-5 mRNA was present at high concentrations in the maternal caruncles and luminal epithelium, and at moderate levels in myometrium. In these regions IGFBP-5 mRNA showed cyclic variations, with concentrations peaking around ovulation, whereas low expression in the endometrial stroma remained constant. During pregnancy, there was additional localization to the endometrial glands; and in all regions, with the exception of the caruncles, concentrations increased significantly with gestational age. In transected uteri, concentrations in the luminal epithelium of the pregnant horn were significantly higher than those in the nonpregnant horn. In the caruncles, IGFBP-5 mRNA formed an intense band just below the tips of the invading fetal villi. Below this band, IGFBP-5 mRNA localized to form a series of rings, which could create a route to allow the fetal villi access into the caruncular stroma for nutrient exchange. In conclusion, IGFBP-5 is abundantly expressed in the ovine reproductive tract, with both the concentration and localization differentially regulated during the cycle and pregnancy.

Expression of insulin-like growth factor binding protein-1 (IGFBP-1) mRNA in the ovine uterus throughout the oestrous cycle and early pregnancy.

Insulin-like growth factors (IGFs) are thought to be important regulators of embryonic and fetal development. The half life, distribution and action of IGFs are modulated by a family of IGF-binding proteins (IGFBP). This study investigated the pattern of IGFBP-1 expression in the ovine uterus during the oestrous cycle and early pregnancy by in situ hybridisation. Uteri were collected from 46 non-pregnant ewes throughout the oestrous cycle and from 12 pregnant ewes between days (D)13 and 22 of gestation. Samples were also obtained on D16-17 from both horns of 5 ewes with unilateral pregnancies following uterine transection. IGFBP-1 expression was quantified as optical density (OD) units from autoradiographs using a Seescan image analysis system. IGFBP-1 mRNA was confined to the luminal epithelium, with a highly significant variation in concentration according to the stage of the cycle. In non-pregnant uteri, IGFBP-1 concentrations were high throughout the late luteal phase and oestrous period, peaking at an OD of 0.76+/-0.119, but concentrations fell below the detection limit (OD<0.01) by D5 before starting to increase again between D7 and 9. During early pregnancy there was no difference in expression between non-pregnant and pregnant ewes on D13 (OD 0.76+/-0.065, n=6 vs 0.71+/-0.070, n=3). As pregnancy progressed there was a significant steady decline in IGFBP-1 expression to 0.04+/-0.02 on D22. In the transected uteri on D16-17, IGFBP-1 mRNA expression was significantly higher in the pregnant than in the non-pregnant horn (0.44+/-0.04 vs 0.10+/-0.02, n=5, P<0.01). In conclusion, the location of the IGFBP-1 suggests that it may play a role in regulating the transfer of IGFs between the endometrium and the uterine lumen. The conceptus may enhance IGFBP-1 expression during early pregnancy. Oestrogen and progesterone may regulate IGFBP-1 expression during the cycle but this requires further investigation.