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S. A. Wood and P. L. Kaye

Summary. When epidermal growth factor (EGF) was added to the medium for culture of preimplantation embryos, morphological development as determined by microscopic observation was unaffected, but 333 nm-EGF stimulated total uptake of [3H]leucine by late morulae/blastocysts which had been cultured for 24 h from morulae. Incorporation of [3H]leucine into protein by these embryos was increased by 0·33, 3·3 and 33 nm-EGF, following a quadratic relationship producing less stimulation at 333 nm, which may indicate down regulation of receptors. The estimated EC50 was ∼0·25 nm. Manipulation of the culture period indicated that the embryos responded to EGF at the morula/blastocyst transition period and immunosurgery was used to show that the increased protein synthesis was restricted to the trophectoderm cells. No mitogenic effect was observed. The effective concentration of EGF is close to that of serum and to values which stimulate other tissues. It is suggested that EGF receptors appear at compaction and that EGF may have a role in differentiation of the trophectoderm cells.

Keywords: EGF; preimplantation embryos; protein synthesis; mouse

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R. G. Glencross, E. C. L. Bleach, S. C. Wood, and P. G. Knight

We reported previously that active immunization of heifers using a synthetic peptide-based inhibin vaccine (bIα(1–29)Tyr30) can enhance ovarian follicular development and ovulation rate during spontaneous oestrous cycles. To extend this study, we investigated the effect of inhibin immunization more closely by monitoring plasma hormone profiles and ovarian activity in bIα(1–29)Tyr30-immunized and control (ovalbumin-immunized) heifers (n = 6 per group) over three consecutive oestrous cycles, which were synchronized and shortened by administering a PGF analogue at intervals of 14 days. Blood samples were collected at 2–8 h intervals for 40 days and the ovaries were examined daily using ultrasonography. Repeated-measures anova showed that inhibin immunization significantly increased plasma FSH concentration (by 52% overall; P < 0.01) and ovulation rate (by 58%; P < 0.01). Both immunized and control heifers showed the same cyclic pattern of plasma FSH (treatment × time interaction; not significant), indicating that the increase in plasma FSH was sustained throughout the cycle. Immunization did not affect the concentration or pattern of secretion of LH, oestradiol or progesterone and had no influence on the timing of the LH surge or ovulation after PG injection. While inhibin immunization increased the number of 'large' (i.e. growing to ≥ 10 mm diameter) follicles that developed during both the preovulatory (by 90%, P< 0.02) and postovulatory (by 190%, P< 0.01) period, there was no difference between groups in the temporal pattern of growth or regression of large follicles or of corpora lutea. These observations confirm a physiological role for ovarian inhibin as a component of the ovarian feedback mechanism controlling FSH secretion in heifers, and support the hypothesis that active immunization of heifers against inhibin enhances ovarian follicular development and ovulation rate by promoting a sustained increase in pituitary FSH secretion.

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G. T. Waites, P. L. Wood, R. A. Walker, and S. C. Bell

Summary. The distribution of α2-PEG, a human analogue of β-lactoglobulin, in endometrium at different phases of the cycle was determined using immunohistochemistry with monoclonal and polyclonal antibodies. In the epithelial cells of glands in the functional zone of the endometrium, α2-PEG was first detectable from Days 19 to 21 during the mid-luteal phase and maximal immunostaining was observed during the end of the late luteal phase. Intense staining in the glandular secretions and weaker staining in surface luminal epithelial cells during this period were observed. A minor population of basal glands contained α2-PEG during the follicular phase. These results suggest that α2-PEG synthesis by the glandular epithelium of the regenerated endometrium is hormonally regulated. Maximal staining occurring during the late luteal phase suggests that regulation may be related to the hormonal requirement for pre-decidualization rather than that required for histologically defined glandular epithelial secretion.

Keywords: endometrium; human; menstrual cycle; pregnancy-associated endometrial α2-globulin; pregnancy proteins

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F. B. P. Wooding, A. P. F. Flint, R. B. Heap, G. Morgan, H. L. Buttle, and I. R. Young

Summary. In the ruminant placenta 15–20% of the trophectodermal epithelium consists of granulated binucleate cells (BNC). In the sheep the granules contain ovine placental lactogen (oPL). These cells migrate from the trophectoderm to form fetomaternal hybrid tissue from implantation to term.

The number of BNC, their percentage migration and the potential secretory activity of the syncytium they form were estimated by semiquantitative transmission electron-microscopical techniques after several surgical techniques and hormone or drug infusions.

BNC numbers decrease normally just before parturition, and this fall could be eliminated by fetal hypophysectomy or induced early by administration of tetracosactrin to intact or hypophysectomized fetuses. If only one twin was treated with tetracosactrin the placenta of the untreated twin did not show the fall in BNC numbers found in the other unless it died in utero some time before sampling. This indicates fetal control of BNC number and migration.

However, fetal catheterization, hypophysectomy, stalk section, adrenalectomy, infusion of mouse epidermal growth factor or bromocriptine had little or no effect on binucleate cell numbers or migration percentages. Maternal carunclectomy, ovariectomy, or epostane or bromocriptine administration also had no consistent significant effect.

Previous reports of degeneration of BNC structure plus a decrease in their number (with bromocriptine) or an increase in migration frequency (after adrenalectomy or stalk section) have not been confirmed by this study. The BNC migration delivers the oPL-containing BNC granules close to the maternal circulation but the variation in migration seems only loosely correlated with the reported maternal oPL concentrations.

The results indicate that BNC migration is independent of the hormonal milieu, but that BNC production is greatly modified by the hormonal changes just before parturition, with cortisol production by the fetus a possible primary cause.

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S McMullen, J C Osgerby, L M Thurston, T S Gadd, P J Wood, D C Wathes, and A E Michael

In the placenta, cortisol is inactivated by NADP+- and NAD+-dependent isoforms of 11β-hydroxysteroid dehydrogenase (11βHSD). Decreased placental 11βHSD 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 11βHSD 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 11βHSD 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 11βHSD 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 11βHSD 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 11βHSD activities.