ABSTRACT

Studies of the binding of ¹²⁵I-labelled epidermal growth factor (EGF) to the ovine placenta were carried out on days 50, 90–100 and 140 of pregnancy. Membrane fractions were purified from the fetal area of the cotyledon. Two classes of binding sites were found. Their dissociation constants (K _d) were not significantly different for the three stages of pregnancy considered (high-affinity K _d 54–70·2 pmol/l; low-affinity K _d 12·2 to 19 nmol/l). However, the number of high-affinity binding sites on days 90–100 was significantly (P < 0·01) greater (146 ± 19 fmol/mg protein) than on either day 50 or day 140 (respectively 74·2 ± 1·26 and 56·3 ± 5·6 fmol/mg protein). Affinity cross-linking studies followed by SDS-PAGE under reducing conditions demonstrated that the major part of the EGF was specifically cross-linked to a protein of molecular weight of 150 kDa and to lesser extent to 180 kDa and 130 kDa proteins. Membranes prepared from unfrozen tissues, in the presence of sodium iodoacetate to reduce endogenous enzymatic conversion of the 180 kDa form to the 150 and 130 kDa forms, still exhibited a major EGF-binding protein of 150 kDa. The occurrence of an increased number of EGF receptors at the period of rapid cotyledonary growth which coincides with the increase in placental hormonal secretions suggests that EGF has a role in the development of the ovine placenta.

Journal of Endocrinology (1993) 136, 43–50

Abstract

The role of IGFs in placental growth is poorly understood. IGF-II receptors have been characterised in the ovine placenta and used extensively for radioreceptor assay, but their evolution during placental development has not been considered. In this study, binding sites for IGF-I were characterised in the ovine cotyledon by binding and cross-linking studies and the evolution of the number of IGF-I and IGF-II receptors on placentae collected on days 50, 75, 100 and 140 of pregnancy were compared. IGF-I bound onto placental membranes with a mean association constant of 1·7 nm ⁻¹ except on day 50 when a lower association constant was observed (0·8 nm ⁻¹). Scatchard analysis of the displacement curves led to a single binding site model. IGF-II was as potent as IGF-I at displacing the binding of ¹²⁵I-labelled IGF-I on those membranes, whereas insulin cross-reaction was only 1%. IGF-II bound on our placental membrane preparations with the characteristics described previously and neither IGF-I nor insulin was able to displace this binding. Affinity cross-linking studies followed by SDS-PAGE under reducing conditions demonstrated that IGF-I was linked to a protein with a molecular weight of about 135 000 Da and IGF-II to a protein of 250 000 Da. The mean ± s.e.m. number of IGF-I receptors was significantly higher on days 50 and 75 than on days 100 and 140 (154 ± 12, 105 ± 11 vs 65 ±4, 48 ±3 fmol/mg, P<0·01). The number of IGF-II receptors followed the same pattern and also showed a decrease towards the end of the pregnancy that was significant (P<0·01) only by day 140 (days 50 and 75, 1 ± 0·08; day 100, 0·75 ±0·04; day 140, 0·51 ± 0·03 pmol/mg). IGF-I receptors were observed in the trophoblast cells of cotyledons removed on day 40 whereas IGF-II receptors were observed in mesodermal cells. These data suggest that IGFs are involved in the placental growth and differentiation processes and that some effects of IGF-II are probably mediated by IGF-I receptors.

Journal of Endocrinology (1995) 144, 179–191

The present study demonstrates that ovine placental lactogen (oPL) (ovine chorionic somatotrophin) may have an important role in the mammogenesis and/or lactogenesis of the ewe. Its effects were compared with that already described for ovine growth hormone (oGH). In the first experiment, 40 nulliparous ewes were induced to lactate by means of a 7 day (days 1-7) oestro-progestative treatment (E2+P4). The ewes from Group 1 (n=12) received no further treatment, while those of the other groups received either recombinant oGH (roGH, 28 micrograms/kg, i.m., twice daily, Group 2, n=12) or recombinant oPL (roPL, 79 micrograms/kg, i.m., twice daily, Group 3, n=12) from day 11 to 20. All ewes received 25 mg hydrocortisone acetate (HC) twice daily on days 18-20. Control Group 00 (n=2) received no steroid treatment at all, and the control Group 0 (n=2) received only the E2+P4 treatment. Thirteen ewes (three from each experimental group and the two of each control group) were slaughtered at the end of hormone treatments (day 21) before any milking stimulus. The 27 remaining ewes from Groups 1-3 were machine-milked and milk yields recorded daily from day 21 to 76. The E2+P4 treatment enhanced the plasma levels of oPRL, oGH and IGF-I between days 1 and 7 by 1.5, 2. 3 and 2.6 times respectively (P=0.002); roGH treatment induced a highly significant enhancement of IGF-I plasma levels from day 11 to 20, whereas a similar effect appeared for roPL-treated ewes only from day 17 to 20 (P<0.01). Eight weeks after the last exogenous hormone injections, milk yields of both roGH- and roPL-treated groups progressively rose to twice that of unsupplemented groups (P<0.001). The mammary DNA content on day 21 was higher for animals which received either oGH or oPL but, due to individual variations in so few samples (n=3), this difference was not significant. No beta-casein was measured in mammary tissue from control ewes, whereas steroid-treated ewes (E2+P4+HC) had higher casein concentrations regardless of subsequent hormonal treatment on days 11-20 (P<0.001). beta-Casein concentrations in mammary parenchyma of roGH-treated ewes did not differ from that of ewes which received only E2+P4+HC; roPL supplementation clearly enhanced expression of beta-casein (P<0.001). IGF-I stimulation by either roGH or roPL was more precisely examined during a second experiment, in which two twice-daily i.m. doses (58 or 116 micrograms/kg) of either roGH or roPL were administered to four groups of six ewes that were E2+P4 treated as those of Experiment 1. A control group (n=6) received no exogenous hormone from day 11 to 13. On day 13, hourly blood samples were taken from all ewes over 11 h. Both doses of roGH significantly stimulated IGF-I in a dose-dependent manner. The 58 micrograms/kg dose of roPL did not significantly stimulate IGF-I, but although being somewhat less efficient than the 58 micrograms/kg dose of roGH, the 116 micrograms/kg dose of roPL significantly stimulated IGF-I secretion (P<0. 001). These results suggest that mammogenesis and/or lactogenesis in the ewe is in part controlled by somatotrophic hormones such as oGH and oPL and that IGF-I could be one of the mediators of these hormones.

In contrast to hypophysectomy, pituitary stalk section in the sheep on any day of the oestrous cycle permits a continued secretion of progesterone which is comparable to that of normal animals (Denamur, Martinet & Short, 1966). These authors postulated that the maintenance of the secretory ability of the corpus luteum after stalk section must be due in part to the continued release of pituitary prolactin. Measurements of plasma prolactin during the normal oestrous cycle of the sheep and after section of the stalk were carried out to verify the hypothesis, and the results are shown in Table 1.

Samples from three normal ewes (E1, E 2 and E 3) were taken daily at 07.00 h from the jugular vein. The onset of oestrus was detected by testing with vasectomized rams six times a day (06.00 h, 07.30 h, 09.00 h, 15.30 h, 17.00 h and 18.30 h). All samples were

We report the ability of sheep placental cotyledonary cells, isolated at different periods of pregnancy (40 to 90 days) to produce ovine chorionic somatomammotrophin (oCS) in in vitro culture conditions. This oCS production increased gradually with stage of pregnancy. Endogenous oCS net production by isolated placental cells was increased, in a dose-dependent manner, by addition of recombinant oCS (roCS). This effect was not observed after addition of recombinant ovine growth hormone. The roCS effect was more potent on cells collected during early pregnancy. Specific immunoprecipitation of oCS revealed that roCS treatment was associated with an increased dose-dependent incorporation of [35S]methionine-[35S]cysteine. These findings provide evidence that oCS may act in a paracrine/autocrine manner to up-regulate its own production during early gestation. We suggest that this autoregulation may be associated with morphological and functional differentiation of the trophoblast during the growth of the placenta.

A specific leptin RIA was developed to assess concentrations of leptin in ovine plasma, and was shown to be efficient with bovine and caprine plasma. A specific, high-affinity antibody was generated against recombinant ovine leptin which, when used in a competitive leptin RIA, provided valid estimates of linearity (r=+0.989-0.998), recovery (102%), repeatability (13%) and limit of sensitivity (0.83 ng/ml for 100 microl sample size). Serial dilutions of five ovine, bovine or caprine plasma samples showed good linearity and parallelism with the recombinant ovine leptin standard curve. A comparison of this RIA was made with a commercial 'multi-species' RIA kit using 56 ovine plasma samples. Major differences were found in assay sensitivity. Non-lactating, non-pregnant, ovariectomized ewes were fed a ration for 65 days which provided 90+/-9% (control; n=12) or 39+/-2% of maintenance energy requirements (underfed; n=16) in order to analyse the respective effects of body fatness (estimated by either an in vivo dilution technique or body condition scoring) and of nutritional status on plasma leptin concentration. There was a significant positive correlation between body fatness or body condition score and plasma leptin levels (r=+0.68, P<0.001 or r=+0.72, P<0.001 respectively). When concentrations of leptin were assessed over time, underfed ewes exhibited a dramatic reduction in plasma leptin values (-56%, P<0.001). These data provide strong evidence that, in sheep, the variations in plasma concentrations of leptin are related to variations in body fatness (35%) and, to a lesser extent, in nutritional status (17%).

SUMMARY

The concentrations of progesterone, oestrogen and luteinizing hormone (LH) in ovarian venous plasma, and of progesterone, follicle-stimulating hormone, LH and prolactin in peripheral plasma were measured in 47 Welsh Mountain sheep at accurately timed stages around oestrus. The histology and ultrastructure of the pre-ovulatory and newly ruptured follicles in these animals were also examined.

The highest ovarian vein levels of oestrogen were invariably obtained from the ovary containing the largest non-atretic follicle. A significant amount of oestrogen was already being secreted by day 13, when both the peripheral and ovarian venous blood progesterone levels were still high.

On day 15 the corpus luteum stopped secreting progesterone; coincident with this decline there was a maximal increase in oestrogen secretion from the largest non-atretic follicle. The thecal cells of this follicle enlarged concomitantly with the rise in oestrogen, and maximal development of the smooth endoplasmic reticulum was observed at or after the oestrogen peak.

By 6 h after the onset of oestrus, progesterone concentration was still very low, and oestrogen values had fallen in most animals, while LH showed a pronounced increase. The maximal level of prolactin was also found at this time.

By 15 h after the onset of oestrus, both progesterone and oestrogen values were low and LH had returned to baseline levels except in a few animals. Ovulation had occurred in 11 out of 12 animals by 30 h after the onset of oestrus and the recently ruptured follicle was just beginning to secrete a little progesterone. Luteinizing hormone and oestrogen values were very low.