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.

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.

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.

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.

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.