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The possibility that prostaglandin E2 (PGE2) has a central action in the fetal lamb arises from studies by Ratter et al. and Robinson. The Prostaglandin E (PGE) release into the fetus may help to preserve fetal growth by decreasing nonessential fetal muscular activity, e.g., fetal breathing movements, thereby sparing glucose. PGE may also maintain a minimum glucose utilization by stimulating insulin release and enhancing pancreatic sensitivity to glucose. PGE2 concentrations in fetal plasma have been shown to increase during late gestation in sheep and this increase parallels the increase in fetal cortisol concentrations. The site of action of PGE2 has also been studied in newborn lambs in which plasma cortisol concentrations increased when PGE2 was infused into the brachiocephalic trunk, but not when infused into the descending aorta. Decreased placental perfusion, caused by placing a clamp on the common iliac artery, results in an increase in fetal cortisol concentrations, which may be secondary to increased fetal PGE2 concentrations.

This study has investigated the effect of continuous intrafetal infusion of PGE2 or saline on hormone concentrations and the length of gestation in sheep. Fetal and maternal vascular catheters were surgically implanted at 112.3 +/- 1.3 days (n = 10), and the infusions were started at 121 +/- 1.2 days of gestation (term = 147). Fetuses were infused with either PGE2 (n = 5; 2 micrograms/min for 48 h and then increased to 4 micrograms/min for the remainder of the experiment) or the vehicle solution (n = 5; sterile isotonic saline) via the fetal carotid artery. In the PGE2-infused group, fetal and maternal plasma PGE2 concentrations increased (P < 0.001) after the change to the higher dose rate (4 micrograms/min) and remained elevated, fetal plasma immunoreactive ACTH (ir-ACTH) concentrations dramatically increased after the start of the infusion being maximal at 11 h before decreasing to match concentrations exhibited by the saline-infused group. Fetal plasma cortisol concentrations increased after the start of the PGE2 infusion (P = 0.05) and increased further after the change to the higher dose rate of 4 micrograms/min (P < 0.001). Concentrations of PGE2, ir-ACTH, and cortisol in the saline-infused group did not change until labor. Plasma concentrations of PGE2 (P < 0.001) and ir-ACTH (P < 0.005) increased on the day of labor in both treatment groups, and fetal cortisol concentrations increased (P < 0.001) in both groups in the last few days before labor. The proportion of low molecular weight ir-ACTH in the plasma of PGE2-infused fetuses was significantly higher than that of saline-infused fetuses (P < 0.001) during the first 15 days of infusion. In the saline-infused group, the proportion of low molecular weight ir-ACTH increased in the last few days before labour (P = 0.001), whereas no change was seen in PGE2-infused fetuses at this time. Maternal plasma progesterone concentrations decreased in both groups in the last few days before labor (P < 0.001). Fetuses infused with PGE2 delivered at 138.4 +/- 2.1 days, whereas control fetuses infused with saline delivered at 148.2 +/- 0.5 days (P < 0.001). The spontaneous increase in PGE2 preceding normal labor may thus play an important role in activation and maturation of the hypothalamic-pituitary-adrenal axis in fetal sheep.

Although it is known that concentrations of immunoreactive ACTH increase during late gestation in fetal sheep plasma, the nature of the ACTH has not been well characterized. We used two-site immunoradiometric assays to separately measure high mol wt ACTH precursors (POMC and pro-ACTH) and ACTH-(1-39) in plasma of fetal sheep with chronic arterial and venous catheters. We compared the ratio of these peptides as a function of gestational age under basal conditions and in response to exogenous vasopressin and/or corticotropin-releasing hormone. Under basal conditions, the concentration of precursors was not changed throughout the last third of gestation; however, ACTH-(1-39) increased significantly approaching term. The molar ratio of precursors to ACTH-(1-39), therefore, decreased from 15.8 +/- 1.0 at 110 days to 7.9 +/- 0.6 at 140 days gestation. At all gestational ages, vasopressin and corticotropin-releasing hormone increased ACTH-(1-39) and precursors, albeit with different time courses. At 120 days gestation, arginine vasopressin plus CRH produced synergistic increases in ACTH-(1-39) and precursors, whereas the response was only additive at other ages. The present results indicate that the elevation in the resting plasma immunoreactive ACTH concentration that occurs near term is constituted by an increase in the concentration of ACTH-(1-39) relative to those of POMC and pro-ACTH, which may have further physiological significance. Also, CRH and AVP are potent stimulators of both ACTH-(1-39) and ACTH precursors.

This study investigated the effects of repeated short term (2-h) intrafetal infusions of prostaglandin E2 (PGE2) on ACTH and cortisol release in fetal sheep during late gestation (119-144 days). We compared the effects of administration of PGE2 (2 micrograms/min) into the fetal carotid artery or jugular vein. PGE2 infusion significantly (P0.001) increased fetal plasma immunoreactive (ir-) ACTH and cortisol concentrations regardless of the vessel used for administration. Saline infusion did not alter the concentrations of ir-ACTH or cortisol for the duration of the experiment. To compare the responses of fetal ir-ACTH and cortisol to repeated intracarotid infusions of PGE2, the hormone data were grouped into five gestational age ranges (119-125, 126-130, 131-135, 136-140, and 141-145 days). Fetal ir-ACTH was stimulated by PGE2 infusion at all gestational ages studied; the greatest response was achieved at the earliest gestational age range, 119-125 days. PGE2 infusion preferentially stimulated the release of low mol wt ACTH [ACTH-(1-39); 60 min from the start of infusion] at all gestational ages (P0.01), but basal low mol wt ACTH did not increase with gestational age until after 140 days. Cortisol concentrations were increased within 30 min of infusion at all gestational ages studied. These results suggest that PGE2 may play a role in maintaining elevated ir-ACTH concentrations in the face of high levels of cortisol in fetal sheep before parturition.

Summary: The major source of PGE2 in ovine pregnancy is the placenta, with secretion occurring bidirectionally into fetal and maternal circulations. The placental output of PGE2 appears to increase when demand on placental function is increased, suggesting that the normally observed increase in its concentration towards term is driven by the growing demands of the fetus. The fetal pituitary is also involved in the control of PGE2 synthesis. PGE2 has potent stimulatory actions on the fetal pituitary to increase both the absolute concentration and the bioactive fraction of ACTH-containing peptides in the fetal circulation. It also directly stimulates glucocorticoid secretion from the fetal adrenal gland. We propose that PGE2 provides a tonic stimulation of the fetal HPA axis in late gestation, contributing to phenomena such as the apparent insensitivity of the pituitary to Cortisol feedback and the increasing sensitivity of the fetal adrenal. Because of its apparent responsiveness to placental workload, it may transduce stimuli from the placenta and transmit them to the fetal HPA axis, giving a possible biochemical basis to the empirically observed correlation between fetal metabolic demand and gestation length.

Parturition in the sheep is preceded by an increase in the plasma concentration of fetal ACTH and an increase in the plasma cortisol concentration. The role and importance of the increase in fetal ACTH in stimulating fetal glucocorticoid synthesis and the subsequent onset of labor require closer examination, as it has been demonstrated that the fetal adrenal becomes more responsive to ACTH in late gestation. This study sets out to determine whether the increase in plasma ACTH in the late gestation fetal sheep is essential for maturation of the fetal adrenal gland and normal delivery. Fetal sheep were either hypophysectomized (HX) and cannulated or cannulated only (intact) at 125 days gestation. Immediately after surgery, HX fetuses were infused with a constant dose of ACTH-(1-24) (ACTH/HX; 100 ng/h.kg, i.v.) or saline (SAL/HX) until uterine electromyography indicated the onset of labor or 161 days gestation was reached (term = 147 +/- 2.6 days). The mean gestational age at labor of the ACTH/HX group was 147 +/- 2.9 days, whereas none of the animals in the SAL/HX entered labor, and they were killed at 161 days gestation. The concentration of ACTH in both ACTH/HX and SAL/HX fetal plasma was less than 2.2 pg/ml throughout the study. The concentration of cortisol in ACTH/HX fetuses mimicked that in intact fetuses in late gestation, reaching 80 ng/ml at term. The concentration of cortisol in SAL/HX fetuses remained less than 5 ng/ml. This study supports the hypothesis that the ovine fetal adrenal becomes increasingly responsive to ACTH in late gestation and indicates that ACTH may only be permissive in the activation of adrenal function. In intact fetal sheep there may be endogenous inhibition of the fetal adrenal, requiring relatively high plasma concentrations of ACTH [100-250 pg/ml ACTH-(1-39)] in late gestation.

Epidermal growth factor (EGF) infused over 3-14 days into fetal sheep of 110-125 days gestation resulted in a number of morphological and endocrine changes. Striking hypertrophy of the skin, wool follicles and their accessory structures was seen, together with a reduction in the ratio of secondary to primary follicles and degenerative changes in wool fibres associated with shedding of fibres. Adrenal, thyroid, liver and kidney weights were increased while thymus weight was decreased. The increase in adrenal size resulted from cortical hypertrophy and was associated with increased cortisol secretion. Thyroid hypertrophy was accompanied by an increase in colloid stores, decreased plasma thyroxine and reverse triiodothyronine (T3) concentrations, unchanged plasma T3 and thyroid-binding globulin and raised thyrotropin (TSH) levels. Thyrotropin receptor affinity and content per gram of tissue were unchanged. Fetal and maternal plasma prolactin and growth hormone levels, and fetal plasma placental lactogen levels, were unchanged, although there was a significant rise in maternal plasma placental lactogen concentrations with high doses of EGF. Other maturational parameters such as switching from fetal to adult haemoglobin and liver glycogen content were unaffected.

The concept that the fetus initiates its own delivery dates from 460 BC, when Hippocrates suggested that, at the appointed hour, the fetus puts its feet on the fundus of the uterus and pushes to effect its own delivery. It was not until the tenth century that the Italian anatomist, Fabricius ab Aquapen-dente, proposed that the chief agent in the initiation of parturition was the muscular action of the uterus. In 1882, Spiegelberg re-emphasized the role of the fetus when he wrote in a German textbook of midwifery that, ‘… the reason why labour occurs at a definite time must be sought for, not in the uterus and its changes, but in the fetus. It is the maturity of the latter that gives the signal.’. In 1933, Malpas reported a series of cases of prolonged pregnancy associated with fetal anencephaly. Malpas concluded that ‘the time of the onset of labour is determined by the foetus’, and ‘the foetal adrenal, pituitary, or nervous system, perhaps in combination, are suggested as tissues possibly concerned in the actual excitation of the neuromuscular expulsive mechanisms’.

The factors involved in the control of steroid secretion from the ovine placenta and in fetal growth are as yet unclear. We hypothesized that factors derived from the fetal pituitary may play a role in the production and release of placental steroids and in growth of the fetus, and have investigated the effects of fetal hypophysectomy performed between day 70 and day 79 of gestation (term = 147 days) on systemic concentrations of hormones derived from the placenta, and on fetal growth. Maternal peripheral progesterone, placental lactogen and uterine vein progesterone increased significantly from day 90 in all ewes. Peripheral concentrations of prostaglandin E2 and peripheral and uterine vein oestrone sulfate increased significantly in the control group but not in the fetal hypophysectomy group. Uterine vein prostaglandin E2 increased significantly after day 95 in the control group and after day 105 in the fetal hypophysectomy group. Early fetal hypophysectomy caused marked growth retardation. The weights of the brain, kidneys and liver of hypophysectomized fetuses were the same as those of controls suggesting that their growth is not under pituitary control. In contrast, the weights of heart and lungs were reduced in proportion to body weight, suggesting that heart, lung and carcass growth were under pituitary control. Our data indicate that the fetal pituitary influences the control of placental steroid and prostaglandin E2 biosynthesis after day 90 of gestation in sheep, but that output of other hormones such as placental lactogen is independent of pituitary control, and may determine organ-specific growth parameters that are unaffected by removal of the fetal pituitary.

Ultrastructural features of the regressing corpus luteum have been studied in the ewe at the end of the estrous cycle. Luteal cells secrete densely-staining granules which have been reported to contain progesterone. This process appears to be maximal at Day 10 of the cycle and begins to taper off noticeably at Day 12. The secretory activity declines gradually till Day 14, but up to this time the fine structure of other cytoplasmic organdIes in the luteal cell shows no remarkable change, save for a gradual increase in the number of autophagocytic bodies which first begin to appear at Day 12. In contrast, on Day 15 there is evidence of marked degeneration; the luteal cells are shrunken, granule secretion has ceased, autophagocytic bodies are numerous and lipid droplets, rarely seen in the fully functional cell, are present in abundance, Lysosomes are observed at all stages of the cycle and there is no dramatic increase in their number towards the end, In some cells at Day 15 the process of degeneration has advanced to the stage where the fine structure of organelles is no longer recognizable and within autophagocytic bodies there are large sites associated with acid phosphatase activity.

The capacity of ovine allantoic fluid to modulate prostaglandin (PG) synthesis was measured in fluid taken from ewes at 100–140 days gestation. An inhibitor of PG synthesis was found in allantoic fluid from ewes at 100–130 days gestation. The inhibitor had the capacity to inhibit PGF2α, PGE2, and 6-keto-PGF1α synthesis by endometrial cotyledon microsomes in a concentration-dependent manner. At 140 days gestation, the inhibitory potency of the fluid was significantly less than that present at 100–130 days gestation, at concentrations greater than 20% (v/v) (p < 0.001). The inhibitor is not albumin and remained active after boiling, dialysis, and protease treatment. It was extracted with chloroform–methanol, indicating that the active portion of the molecule is a lipid. We suggest that parturition in the sheep may be associated with a local withdrawal of inhibition of prostaglandin synthesis.

Oxytocin (OT), progesterone and prostaglandin F2α (PGF2α) concentrations were measured in the utero-ovarian vein (UOV) of ewes which displayed persistence of the corpus luteum (CL). During the period of expected luteolysis, the frequency of OT and PGF2α pulses in the UOV was significantly (P < 0.005 for both) lower in ewes with persistent CLs, compared with ewes that underwent normal luteal regression. In contrast, the amplitude of both OT and PGF2α pulses was similar in both groups of animals. It is suggested that persistence of the CL resulted from a decreased PGF2α pulse frequency, which may have arisen from a decreased frequency of stimulation by OT. In two persistent CL ewes, however, it appeared that a failure at the level of the uterus may have contributed to the observed decrease in PGF2α release. Although a PGF2α analogue (Lutalyse) infusion into the uterine vein of two ewes with persistent CLs failed to induced luteolysis, it did stimulate a large release of OT into the UOV. This suggests that persistent CLs maybe more resistant to PGF2α and, that at day 22 post-oestrus, these CLs are capable of releasing large quantities of OT into the UOV.

The effectiveness of trilostane and azastene as inhibitors of adrenal steroidogenesis was compared by in-vitro and in-vivo methods. A radioimmunoassay was developed for the measurement of cortisol in ovine plasma, incubation medium and tissue extract using a specific antiserum raised against cortisol 21-acetate,3-carboxymethyloxime : bovine serum albu Trilostane (20 μmol/l) decreased cortisol synthesis and release both in unstimulated and in ACTH-stimulated adrenal tissues in vitro. The same concentration of azastene had a lesser effect on unstimulated adrenals and was completely ineffective in blocking the stimulatory action of ACTH. In vivo, trilostane suppressed adrenal steroidogenesis in pregnant and cyclic ewes but the suppression in pregnant ewes was over a longer period, and after lower doses. It is concluded that trilostane had an inhibitory effect on ovine adrenal steroidogenesis both in vitro and in vivo.

Premature induction of delivery in fetuses infused with graded doses of cortisol was brought about in 123.5 +/- 7.7 h (mean +/- SEM, n = 6) after the start of cortisol infusion. This treatment caused a rise in fetal plasma cortisol similar to that observed at normal delivery. Maternal and fetal progesterone and 20 alpha-dihydroprogesterone concentrations decreased to basal levels during infusion of cortisol to the fetus. Induction of premature delivery was delayed or prevented by concomitant treatment of the ewe with progestagen. Maternal intramuscular injection of 100 mg progesterone, 2 times daily, prevented delivery in four of four ewes treated during the time that cortisol was infused into the fetus (11-13 days). Maternal plasma progesterone and 20 alpha-dihydroprogesterone concentrations were maintained during this period, but fetal plasma progesterone concentrations decreased to the same extent as in the fetuses infused with cortisol alone. A single intramuscular injection of 250 mg of medroxyprogesterone acetate to ewes on the day before commencement of infusion of cortisol to the fetus prevented delivery in four of six ewes during the time that cortisol was infused for 9, 13, 14, and 15 days, respectively. One ewe delivered a live lamb at 133.5 h and another at 147.7 h after the start of infusion of cortisol to the fetus. Maternal and fetal plasma cortisol, progesterone, and 20 alpha-dihydroprogesterone concentrations were similar to those observed during infusion of cortisol alone to the fetus. Although fetal cortisol concentrations rose in a similar fashion, and to a similar extent, in all three groups during infusion of cortisol to the fetus, fetal 11-desoxycortisol concentrations only rose above basal levels close to the time of delivery in cortisol-infused fetuses or, in the progestagen-treated groups, when the fetus showed signs of being stressed.

Synacthen has been infused at 10 μg/h into 4 hypophysectomized foetal sheep. This caused a rise in adrenocorticotrophin to a mean value of 512 pg/ml which remained fairly constant and did not show the large fluctuations seen on infusion into intact foetuses. The Synacthen half-life in the circulation had a mean value of 0.27 min. Hypophysectomy did not necessarily delay the foetal corticosteroid response to Synacthen infusion. The corticosteroid concentration achieved had a mean value of about 50 ng/ml which is substantially below that for intact foetuses and in one case it was maintained at only 20 ng/ml. Despite this delivery occurred within 5 days. Dexamethasone infusion reduced the plasma adrenocorticotrophin concentration by about 70 % and the plasma cortisol concentration by about 60 %.

To determine whether the adrenal gland of the bandicoot is actively producing steroid hormones at birth, the cellular structure was examined by electron microscopy, and the concentration of cortisol in the gland was determined by radioimmunoassay. Two distinct cell populations were seen in the adrenal gland. One group of cells possessed dark staining granules, normally observed in catecholamine-secreting cells, and the second group contained large amounts of smooth endoplasmic reticulum and mitochondria with tubulo-vesicular cristae. Both features are characteristic of steroid-secreting cells. A concentration of 0.094 ng of cortisol per adrenal was determined by pooling glands from nine individual newborn bandicoots. This evidence of cortisol secretion from the bandicoot adrenal at birth indicates that, as in many eutherians, the marsupial adrenal may play an important role in determining the length of gestation.

SUMMARY Progesterone concentrations in the peripheral plasma of cows were measured by a protein-binding radioassay method. The mean concentration was lowest at oestrus (0·44 ng/ml) and then increased to a maximum of 6·8 ng/ml about day 14 of the 21-day cycle. The concentration decreased rapidly during the last 4 days of the cycle, reaching low levels on the day before oestrus. There were no significant changes in progesterone concentration during oestrus. After ovariectomy the plasma progesterone concentration decreased to a very low level (< 0·4 ng/ml). After hysterectomy, progesterone concentrations remained high for longer than in a normal cycle. At puberty, plasma progesterone concentrations indicated cyclic ovarian activity before the first observed oestrus. Daily treatment of cows with oxytocin (0·4 u./kg body weight) from day 2 reduced the oestrous cycle length to 9 days, but did not significantly alter the slow increase in plasma progesterone concentration during the first 5 days of the cycle. Plasma progesterone concentrations decreased again after day 5 to low values. Plasma progesterone concentration during early pregnancy was similar to the luteal phase value (4–6 ng/ml), declined during mid-pregnancy and then increased to a maximum (7–8 ng/ml) at about 240 days gestation. The concentration declined 2–3 weeks before calving. During lactation progesterone concentrations were very low until the resumption of cyclic ovarian activity. The first post-partum cycle, whether accompanied by observed oestrus or not, was usually preceded by a small increase in plasma progesterone concentration 3–5 days before the start of the cycle. Undernutrition significantly increased plasma progesterone concentrations in mid- and late pregnancy. Undernutrition of non-pregnant cows increased progesterone concentrations during the luteal phase of the first cycle, but reduced it in later cycles.

SUMMARY Progesterone concentrations in the peripheral plasma of goats were measured by a protein-binding assay. The mean concentration was extremely low on the day of oestrus (0·2 ng/ml) and was not significantly different from that found in anoestrous or ovariectomized animals. The concentration increased to a maximum of 4 ng/ml on about day 10 of the 21-day cycle, and decreased rapidly during the last 3 days of the cycle. Plasma progesterone concentration during early pregnancy (2·5–3·5 ng/ml) was similar to the luteal phase value and remained steady from day 8 to day 60. Between days 60 and 70 there was a secondary increase in progesterone concentration which was maintained at this increased level (4·5–5·5 ng/ml) until just before parturition. In twin-bearing animals, the secondary increase was greater. Progesterone concentration decreased rapidly during the 1–2 days preceding parturition, but the concentration was still quite high on the day of parturition (1·25 ng/ml). The progesterone concentration in peripheral plasma was markedly increased during anaesthesia and the operation. After bilateral ovariectomy of the pregnant goat, peripheral progesterone concentration fell rapidly from 9 to 2·5 ng/ml during the first ½ h and then more slowly during the next 5–6 h. The animals aborted 36–48 h later. A consistent positive arterio—venous difference for progesterone was observed across the pregnant uterus in two unanaesthetized goats. These results indicate that the ovary is the main site of progesterone production in the pregnant goat and that production by the placenta is small and unlikely to influence the level of this hormone in the maternal circulation.

THE pituitary hormone adrenocorticotrophin (ACTH) belongs to a ‘family’ of peptides derived from a common ‘stem hormone’ (Fig. 1). Depending on the way in which the stem hormone is cleaved, the various peptides are expressed and it is possible that qualitative changes in the expression of the peptide ‘family tree’ may be responsible for the alteration in fetal adrenal function which precedes birth1. We now report evidence for such changes in the rhesus monkey, which has a fetal adrenal comparable with that of man.

Serial, rapid measurements of left ventricular myocardial blood flow in trained, unanesthetized dogs have been made by injecting krypton 85 through chronically implanted coronary artery catheters and counting with an external scintillation detector. Precordial radioactivity declined as a single exponential function during the first 2 min after injection, suggesting a single rate of myocardial blood flow. Simultaneous estimations with Kr85 and blood flowmeters in acute experiments established the accuracy and reproducibility of the technique. Myocardial blood flows between 40 and 55 ml/100 g/min were observed repeatedly in three well-trained, unanesthetized dogs in the basal state.

The granulosa luteal cells of the sheep corpus luteum secrete their hormonal products by exocytosis of granules. Electron micrographs of randomly selected granulosa cells from nine corpora lutea at day 10 of the oestrous cycle were examined to obtain the cellular density of these granules. From the area of the cell, calculated using an x-y digitiser, and the number of granules observed, the number of intracellular secretory granules per microns 3 of luteal cell cytoplasm was calculated. There was a large variation in the number of granules per cell within the same corpus luteum and between corpora lutea taken at the same stage of the cycle. The number of intracellular granules in nine corpora lutea varied from 2.12 +/- 1.05 granules per microns 3 (mean, SD, n = 30) cells to 0.36 +/- 0.18 granules per microns 3 (mean, SD, n = 26). These morphological data suggest that the variation in granule synthesis in individual cells may contribute to the variation in hormone content of corpora lutea at the same stage of the cycle, and the episodic release of hormone into the plasma.

Cells from adrenal glands of 140–160 days foetal, neonatal and infant Rhesus monkeys (Macaca mulatta) were prepared, by collagenase digestion and incubated with 10 pg– 16 ng/ml ACTH. The production of cortisol, androstenedione, corticosterone, progesterone and prostaglandins was measured. The cortisol production in the 140 day and 160 day foetuses and in the neonatal adrenal cells was comparable. It was 2-fold higher in adrenal cells of 6 month infant monkeys. In all the groups there was an increasing production of cortisol with increasing ACTH concentration, and a response to low physiological concentrations of ACTH. The androstenedione production was significantly greater in the 160 day foetuses than in either those of 140 days or of the neonate which demonstrated a poor response to increasing ACTH concentrations. It responded well to increasing ACTH in adrenal cells from 6 month infant monkeys. Corticosterone output was 1/10th of cortisol with only the 140 day foetuses showing an increase in production with increasing ACTH concentrations. The results demonstrate that cells of the primate foetal adrenal gland are not inherently unresponsive to ACTH stimulation as regards cortisol production, which per/μg DNA does not appear to change over the last 25 days before term.

SUMMARY Radioreceptor assays for prolactin-like (lactogenic) activity and growth hormone (GH)-like activity have been used to study concentrations of caprine placental lactogen (PL) in the circulation during pregnancy. Both lactogenic and GH-like activities increased from less than 100 ng/ml (ovine prolactin- and human GH-equivalents) about 60 days after mating to reach peak levels (400–1600 ng/ml) between days 110 and 130 of pregnancy. The levels of both activities increased in essentially the same fashion but during the last 15 days of pregnancy, lactogenic activity declined less than GH-like activity. This divergence was most pronounced at parturition when levels of lactogenic activity increased (∼ 700 ng/ml) despite very low (< 200 ng/ml) levels of GH-like activity being measured and this probably reflected increased secretion of pituitary prolactin near parturition. When serum from a pregnant goat or a simple alkaline extract of placental cotyledons was fractionated on a column packed with Sephadex G-100, lactogenic and GH-like activities eluted together with distribution coefficients of approximately 0·5–0·6. The possibility that caprine PL serves physiologically as a luteotrophin and/or mammotrophin during pregnancy in goats is discussed.

Nuffield Institute for Medical Research and Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Headington, Oxford, 0X3 9DU (Received 6 April 1978) Growth hormone (GH) has been located in the ovine foetal pituitary gland by day 50 of gestation (Stokes & Boda, 1968). The concentration of GH in the plasma of foetal sheep is ten times higher than the postnatal value, increasing from 40 ng/ml on day 100 of gestation to 100–120 ng/ml on day 140 (Bassett, Thorburn & Wallace, 1970). After foetal hypophysectomy, the concentration of GH falls to < 2 ng/ml, indicating that it originates in the foetal pituitary gland (Wallace, Stacey & Thorburn, 1973). Labelled GH does not cross the ovine placenta (Wallace et al. 1973). After sectioning the foetal pituitary stalk, the concentration of GH in the foetal plasma drops to approximately 5 ng/ml (Wallace et al. 1973), which implies that the secretion of GH

Pregnenolone sulphate, pregnenolone, progesterone and 20α-hydroxy-4-pregnen-3-one concentrations in peripheral plasma of normal cyclic ewes were measured by radioimmunoassay. The concentrations of these steroids were correlated with that of progesterone. The concentrations of all the steroids measured in peripheral plasma varied in a cyclic manner and showed a significant (P Peripheral plasma concentrations of these steroids in ovariectomized and ovariectomized, dexamethasone-treated ewes were also determined. The plasma concentration of progesterone in ovariectomized ewes was undetectable but the concentrations of pregnenolone sulphate, pregnenolone and 20α-hydroxy-4-pregnen-3-one remained similar to those observed at oestrus. Administration of dexamethasone to ovariectomized ewes had no effect on pregnenolone sulphate or pregnenolone concentrations but 20α-hydroxy-4-pregnen-3-one concentrations, which were already very low, decreased further. It is proposed that the ovary, probably the corpus luteum, secretes pregnenolone sulphate, pregnenolone and 20α-hydroxy-4-pregnen-3-one; however, pregnenolone sulphate and 20α-hydroxy-4-pregnen-3-one may also arise from the metabolism of circulating pregnenolone and progesterone. J. Endocr. (1987) 113, 231–237.

Synacthen (10 μg/h) infused into foetal sheep at about day 125 of gestation caused premature delivery within 4 days. During the first 24 h of the infusion the secretion of adrenocorticotrophin was depressed and the assayable hormone concentration was within 500–1000 pg/ml. Subsequently, large fluctuations occurred in the adrenocorticotrophin concentration in the pasma of foetal sheep which were consistent with the stimulation of adrenocorticotrophin secretion. No transfer of Synacthen to non-infused twin foetuses was observed. The plasma corticosteroid concentration in the infused foetus also showed a small rise during the first 24 h of the infusion followed by large fluctuations in concentrations that coincided with the changes in adrenocorticotrophin. No consistent changes in the plasma corticosteroid concentration in the non-infused twin foetus were observed. These results are discussed in relation to the hormone changes occurring during normal delivery.

The concentration of progesterone in maternal plasma of the bandicoot increased from 3.9 to 12.6 ng/ml between 11 and 4 days before parturition and remained at this level for at least 7 days. Concentrations of 13,14-dihydro-15-keto-prostaglandin F-2 alpha increased from basal levels of 288 pg/ml (4 days before birth) to maximal levels of 2534 pg/ml immediately after parturition, then decreased progressively. It is suggested that PGF-2 alplha, but not progesterone, influences the initiation of parturition in this species.

Summary The effect of gestational age on the synthesis of prostaglandins (PG) by isolated preparations of uninucleate and binucleate cells of the ovine placentome has been investigated. PG synthesis by the cells was dependent upon cell number in a linear manner, and was significantly inhibited by indomethacin, but not affected by the addition of exogenous arachidonic acid. The net output of PG by the cells increased progressively with increasing gestational age of the ewe from 35 to 145 days, particularly after 100 days' gestation. The cells prepared from tissue obtained during the last trimester of pregnancy have the ability to produce PGE 2 and PGF 2 α in approximately equal amounts, and a lower synthetic capacity for 6-keto PGF 2 α . The cells also have the capacity to metabolize PGF 2 α to 13,14-dehydro, 15-keto PGF 2 α . Thus the fetal trophoblast cells appear to be the major site of PG production by the placenta at parturition.

Epidermal growth factor-like activity has been detected by radioreceptor assay and radioimmunoassay in the developing chick embryo. Very little activity could be detected prior to Day 8 of embryonic life (hatching is at Day 21). A peak of EGF activity was detectable by both assays over Days 10 to 12. The EGF activity then fell to virtually undetectable levels during Days 14 to 17. A later rise in RRA detectable EGF like activity was then observed over Days 18–20. The EGF activity from a Day 11 embryo chromatographed on high-performance liquid chromatography as a single peak, with very high recovery of activity, at a later elution position than mouse EGF or human EGF.

Hyperglycaemia was produced in chronically catheterized fetal lambs and pregnant ewes by the infusion of glucose into the fetus. Plasma concentrations of placental lactogen did not change significantly in either fetal or maternal circulations. Fetal and maternal hypoglycaemia was induced by administration of insulin to the fetus and ewe separately. Plasma concentrations of placental lactogen in the fetus did not change significantly but maternal plasma concentrations fell slightly after hypoglycaemia in either fetus or ewe. Plasma concentrations of placental lactogen rose in both the ewe and fetus during prolonged fasting of the ewe. These results neither confirm nor refute a role for placental lactogen in intermediary metabolism of the pregnant ewe and fetus but glucose concentration alone is unlikely to be a significant factor in the control of secretion of this hormone.

The concentration of prolactin in samples of fetal plasma and amniotic fluid collected from sheep with indwelling catheters has been measured by radioimmunoassay. There was considerable variation between animals in the concentration of prolactin in fetal plasma which ranged from 50 μg/l. In five out of eight fetuses which were sampled at intervals throughout the last 30 days of pregnancy, there was an increase in the concentration of prolactin in the plasma before parturition. The concentration of prolactin in amniotic fluid was consistently low (approximately 1 μg/l). Induction of premature parturition by intrafetal infusion of dexamethasone was accompanied by an increase in the concentration of prolactin in only one of three fetuses infused. These results indicated that prolactin is present in fetal plasma but that an increase in prolactin in fetal plasma before the onset of parturition does not always occur. The low concentration of prolactin in amniotic fluid contrasts with the high concentrations of this hormone found in the amniotic fluid of the primate.

SUMMARY A radioimmunoassay technique for measuring the thyrotrophin (TSH) concentration of sheep and cattle plasma is described. The sensitivity of the assay allowed the measurement of 1–50 ng TSH/ml unextracted plasma. Cross-reaction with ovine luteinizing hormone, prolactin and growth hormone was very low. The average recovery of added TSH was 103 ± 4·1 (s.e.m.)% and the between-assay coefficient of variation was 13·8%. The normal plasma TSH levels of sheep and cattle were approximately 2·5 ng/ml (5 mu. bovine TSH/100 ml). Foetal sheep had plasma TSH concentrations of approximately 3·2 ng/ml during the last 20 days of gestation. Levels of TSH in the circulation decreased abruptly after hypophysectomy of the foetal lamb and a decline in the plasma thyroxine (T4) concentrations was also apparent within 24 h of the operation. However, thyroidectomy of adult and foetal sheep did not increase plasma TSH concentrations until almost all the T4 had been cleared from the circulation. The injection of T4 into thyroidectomized sheep rapidly reduced plasma TSH concentrations to normal values. However, the continued injection of T4 did not further reduce TSH concentration. The injection of T4 or triiodothyronine into normal sheep was also without effect on plasma TSH concentrations.

The mechanism by which prostaglandin F2α terminates luteal function in the sheep is unclear even though it is used extensively in animal husbandry. At the time of luteal regression, a decrease in 3β-hydroxysteroid dehydrogenase (3β-HSD) activity is apparent in the corpus luteum, but it is not known whether the decrease in enzyme activity is the primary cause of structural luteolysis. The effect of trilostane, a 3β-HSD inhibitor, on luteal function and morphology has therefore been investigated. Intravenous injection of trilostane in the mid-luteal phase of the oestrous cycle caused a decrease in ovarian tissue progesterone content. A transient decrease in peripheral and utero-ovarian vein plasma progesterone was observed but there was no significant effect on the length of the luteal phase of the cycle. There was no significant change in plasma 13,14-dihydro-15-oxo-prostaglandin F2α during the period when plasma progesterone was depressed. Morphological examination of the corpora lutea revealed a decrease in the concentration of electron-dense granules without any other features of impending luteal regression. When plasma progesterone was reduced for more than 10 h by two injections of trilostane 4 h apart, there was again no subsequent effect on the length of the oestrous cycle or on the return to oestrus. Plasma progesterone returned to preinjection levels within 24 h of injection. This evidence suggests that competitive inhibition of 3β-HSD activity, per se, is ineffective in bringing about structural luteolysis. J. Endocr. (1984) 100, 61–66

Oxytocin, oxytocin-associated neurophysin (neurophysin), prostaglandin F2 alpha (PGF2 alpha), and progesterone concentrations were measured in the utero-ovarian vein (UOV) of sheep during the estrous cycle and early pregnancy. On days 13-16 of the cycle, large pulses of PGF2 alpha, oxytocin, and neurophysin were measured in samples collected at hourly intervals from the UOV draining a corpus luteum (UOV/CL). Most of the PGF2 alpha pulses (96.5%) coincided with a pulse of both oxytocin and neurophysin, whereas only 55.6% of oxytocin pulses coincided with a pulse of PGF2 alpha. Therefore, during luteolysis in sheep, uterine PGF2 alpha release is closely associated with ovarian oxytocin release, and oxytocin release is unlikely to be dependent upon a uterine PGF2 alpha stimulus. During frequent sampling, coincident oxytocin pulses were measured in 1) both UOVs when a CL was present in both ovaries and 2) the jugular vein, carotid artery, and UOV/CL, with a significantly higher oxytocin pulse concentration occurring in jugular venous compared with carotid arterial plasma. Pituitary and luteal release of oxytocin may, therefore, occur simultaneously and be controlled by a circulating factor in sheep. Compared to days 13-16 of the cycle, significantly (P less than 0.001) fewer pulses of PGF2 alpha, which were significantly (P less than 0.001) smaller in amplitude, were measured in UOV samples collected frequently during early pregnancy. The frequency of oxytocin pulses observed in the UOV/CL of pregnant sheep was not significantly (P greater than 0.1) different from that observed in cyclic ewes, although most (86.4%) oxytocin pulses occurred in the absence of a PGF2 alpha pulse. In contrast, when a pulse of PGF2 alpha was observed in the UOV/CL of pregnant ewes, it usually coincided with a pulse of oxytocin. The suppression of uterine PGF2 alpha release during early pregnancy is not considered to result from a lack of stimulation by oxytocin.

The affinity of danazol for oestrogen, androgen and progesterone receptors in human endometrium and myometrium was determined, to study the mechanism of action of this drug in the treatment of endometriosis. The ability of danazol to combine with each of the three types of receptor was similar in both endometrium and myometrium. The capacity of danazol to compete with oestradiol-17 beta for the oestrogen receptor was very low (1.72 +/- 0.48 X 10(-3%) cross reaction, mean +/- SEM) and danazol, at the maximum concentration used, was unable to saturate the receptor; but danazol's ability to compete with progesterone for its receptor was considerably higher (8.41 +/- 1.65% using progesterone, 1.95 +/- 0.41% using R5020) and was saturable. Danazol was also able to displace dihydrotestosterone from the cytosol androgen receptor (6.29 +/- 1.82% cross reaction). The association constant of oestradiol for the endometrial and myometrial oestrogen receptors was 2.19 X 10(9)M-1 and 7.45 X 10(9)M-1 respectively, while that of progesterone and dihydrotestosterone for their receptors was similar in endometrium and myometrium (mean 0.25 +/- 0.06 X 10(9) M-1 and 3.62 +/- 1.67 X 10(9) M-1 respectively). Using R5020, the association constant for the myometrial progesterone receptor was 2.50 +/- 0.73 X 10(9) M-1. We conclude that, in view of the high circulating levels of danazol present in patients being treated for endometriosis, it is possible that danazol may bind to, and partly saturate, endometrial and myometrial oestrogen, progesterone and androgen receptors during treatment. An explanation may thus be provided for some of the diverse actions of this drug.

The factors responsible for the growth of the fetal adrenal, and its increase in cortisol secretion during late pregnancy have been examined in experimental studies in sheep. The fetal pituitary is necessary for these changes to occur. However, the increase in cortisol secretion at term is not preceded by an increase in ACTH in fetal plasma. Prolactin increases in parallel with fetal cortisol. ACTH is elevated in fetal plasma during late pregnancy, despite the demonstration of a negative feedback relationship with cortisol, suggesting an overriding stimulus to ACTH. Comparison of fetal ACTH levels during Synacthen (β1–24 ACTH) infusion into intact and hypophysectomized fetal lambs demonstrates endogenous ACTH release at this time. The possible mechanisms by which negative feedback may be overridden are discussed. Studies in vivo and in vitro show that there is a maturation of fetal adrenal sensitivity during late pregnancy. In vivo at days 120–130, ACTH provokes little or no increase in the fetal adrenal secretion of cortisol, whereas intra-fetal infusion of prostaglandin E2, but not PGF2α, provokes a rapid 300% increase in the cortisol concentration in fetal plasma. These findings indicate that the fetal adrenal has the pathway necessary for cortisol production by day 130 p.c., and suggest that adrenal maturation is a key factor in the release of cortisol which precedes parturition.

Groups of three goats at 50, 90 and 130 days of gestation were passively immunized against ovine LH (oLH) by i.v. infusion of 8 ml serum equivalent of the immunoglobulin fraction of rabbit anti-oLH serum (LHAS). Goats at the same stages of gestation as above served as controls and received 8 ml serum equivalent of the immunoglobulin fraction of normal rabbit serum (NRS). Plasma concentrations of progesterone were determined by specific radioimmunoassay of blood collected at 20-min intervals from 6 h before infusion of LHAS or NRS to 12 h after infusion. Less frequent sampling was performed from 2 days before to 6 days after infusion. Plasma from all LHAS-immunized goats exhibited binding of oLH. Twelve hours after immunization, titres ranged from 1:135 to 1:215. All LHAS-treated goats had titres of less than 1:10 by 5 days after immunization, but a low level of oLH binding was still detectable. Treatment with LHAS or NRS did not shorten the length of gestation, with all goats delivering live offspring between 142 and 147 days after conception. Plasma concentrations of LH ranged from less than 0·15 μg/l to 4·8 μg/l and were greater than 0·15 μg/l in 181 of 255 samples (71%) for both the NRS-treated group, throughout the experiment, and the LHAS-treated groups before infusion of antiserum. Luteinizing hormone was not detectable in plasma samples obtained after LHAS infusion in goats at 50 or 130 days of pregnancy. Plasma concentrations of LH exceeded 0·15 μg/l in only five of 51 (10%) samples in 90-day-pregnant goats treated with LHAS, the maximum value reached being 0·80 μg/l. Plasma concentrations of progesterone did not alter significantly after infusion of LHAS or NRS at 50, 90 or 130 days of gestation, nor were they significantly different between the groups treated with LHAS and NRS. The role of LH in the maintenance of pregnancy in the goat after 50 days of gestation is questioned. J. Endocr. (1987) 114, 413–436

A single injection (100 mug i.m.) of Estrumate (I.C.I. 80996) was used to induce luteal regression on day 8 of the estrous cycle in 3 sheep. Progesterone levels in the utero-ovarian vein and femoral artery had fallen within 6 h to less than 50% of the concentrations seen before injection of the analogue. Luteolysis was not associated with endogenous production of PGF. The concentration of PGF in the uteroovarian vein began to increase 27-39 h after the administration of Estrumate, reaching a mean maximum concentration of 1455pg/ml 48 h after Estrumate. The mean concentration of PGF in the utero-ovarian vein between 36-69 h after Estrumate was significantly greater than during the 24 h before Estrumate (control period) or during the 0-30 h immediately after injection (both P less than 0.001). The maximum secretion of estradiol and the pre-ovulatory LH peak occurred during the period of elevated PGF concentrations in the utero-ovarian veins. The possible importance of endogenous PGF production at this time is discussed.

A technique for implanting and maintaining catheters in both utero-ovarian veins (UOV) of intact, conscious sheep has been developed. Both UOV catheters remained patent for more than 2 weeks in 72.7% of animals. In non-pregnant ewes, large pulses of PGF2α were observed in UOV plasma collected on days 13 to 16 of the cycle (frequency 1.3 ± 0.2/12 h; pulse height range 1.0–26.9 ng/ml). In contrast, on days 13 to 16 of gestation, significantly (P < 0.001) fewer pulses of PGF2α (frequency 0.42 ± 0.2/12 h) were observed in both UOVs of pregnant ewes, which were significantly (P < 0.001) smaller in amplitude (pulse height range 1.4–4.9 ng/ml). The maintenance of the corpus luteum (CL) during early pregnancy may therefore directly result from a decreased uterine PGF2α release. During the luteal phase (day 4–12), the PO2 of UOV blood draining an ovary containing a CL (UOV/CL) was significantly higher than blood in the contralateral UOV (P 2 of UOV/CL blood had decreased and was not significantly different to that observed in the contralateral UOV (P >0.1). On day 17, the PO2 of blood in both UOVs was elevated significantly compared to levels observed on the two preceding and the two following days (P < 0.001). We consider that the higher PO2 in UOV/CL blood resulted from a very high luteal blood flow, and that the decrease on days 15 and 16 of the cycle resulted from a decline in luteal blood flow during luteolysis. The subsequent increase in PO2 of blood from both UOVs on day 17 was considered to result from an increased uterine blood flow.

The debate about the relative importance of mother and fetus in the initiation of parturition can be traced back to as early as 460 BC. At this time, Hippocrates proposed that the fetus initiated its own delivery by placing both feet on the fundus of the uterus and pushing. Many years later, the great Italian anatomist, Fabricius ab Aquapendente, argued that the mother played the primary role in initiating delivery of the baby and this was achieved by the contraction of the uterine muscle.

SUMMARY The progesterone concentration in the peripheral plasma of ewes throughout pregnancy has been determined by a protein-binding method. Plasma progesterone concentrations during the first 50 days of pregnancy (2–3 ng./ml.) were not significantly higher than peak concentrations during the luteal phase in cycling non-pregnant ewes, but there was no decrease in the concentration 15–20 days after mating as occurs in non-pregnant ewes. Between 50 and 120 days after mating the plasma progesterone concentration increased steadily to values 2–5 times that found in early pregnancy. These high concentrations were maintained until lambing. A decrease in progesterone concentration during the week preceding lambing was usually, but not always, observed. Mean plasma progesterone concentrations during the last 50 days of pregnancy in ewes with twins were approximately twice those in ewes with a single foetus.

SUMMARY The rate of removal of immunoreactive, intravenously injected 125I-labelled sheep growth hormone (GH) was used to calculate metabolic clearance rates (MCR) in two foetal lambs at 130 days of gestation and in two 6-day-old lambs. The mean MCR calculated for the foetuses was 2·9 ml/min/kg and for the lambs 3·1 ml/min/kg. The concentration of GH in plasma sampled before injection was determined immunologically and the values were used to calculate production rates. A production rate of 924 ng GH/min was calculated for the foetuses and 85 ng GH/min for the lambs. The effect of sectioning the pituitary stalk was studied in two foetuses; after the operation there was a rapid decrease in the circulating levels of GH. Hypophysectomy in two other foetuses also caused an abrupt decrease in plasma GH concentration. It was concluded from these experiments that the exceptionally high concentrations of GH in the plasma of foetal lambs could not be attributed to impaired removal of the hormone from the circulation. The direct cause of the increased hormone concentrations was a high rate of GH secretion resulting from active stimulation of the foetal pituitary by the hypothalamus.

SUMMARY Intravenous infusions of glucose into lambs in utero (130–150 days) and after birth, confirmed the marked post-natal increase in the magnitude of the response of plasma insulin to glucose. These studies also suggest that insulin secretion in foetal lambs is stimulated by glucose at lower plasma concentrations than in lambs after birth. The short-chain fatty acid, valeric acid, given as the sodium salt, caused a very rapid increase in the plasma insulin level of foetal lambs, when given either by intravenous injection or infusion. When birth was induced after only 135 days of gestation by i.v. infusion of a synthetic adrenocorticotrophin preparation (Synacthen) into foetal lambs there was also a prematurely induced maturation of the insulin secretory response to glucose. In these prematurely born lambs the insulin secretory response to i.v. glucose infusion was similar to that of normal lambs after birth and differed greatly from that of normal foetuses of similar age. The results indicate that maturation of the insulin secretory mechanism in the lamb is associated with parturition and suggest that these changes may be consequences of the increasing corticosteroid secretion in the foetus during the last few days of gestation.

SUMMARY The ovarian and part of the uterine venous drainage was diverted through the superficial anterior mammary vein leaving the ovary in its normal position and preserving utero—ovarian relationships. The operation has been performed successfully in 18 pregnant Merino ewes (70–120 days' gestation). Blood samples were collected by direct puncture of the mammary and jugular veins during the latter half of pregnancy and the subsequent breeding season. All ewes lambed normally after a gestation period of 146–151 days. Twelve of the 16 surviving ewes resumed cyclic oestrous behaviour. Plasma progesterone concentration was measured by a competitive proteinbinding assay. Utero—ovarian venous blood flow and rates of ovarian progesterone secretion were measured throughout the oestrous cycle. Progesterone secretion during the luteal phase of the cycle was in the range of 4·5–6·0 mg/day. The progesterone concentrations in peripheral plasma were similar to those found in normal ewes.

SUMMARY Using a protein-binding technique, progesterone concentrations in peripheral plasma (jugular vein) were measured throughout the oestrous cycle of 24 ewes. Examination of the specificity of the method by thin-layer chromatography indicated that interference from other steroids was not significant in sheep plasma. During the first 4 days of the cycle (days 0–3), plasma progesterone concentrations were below 0·4 ng./ml., increasing to a mean level of 1·5–2·5 ng./ml. between days 4 and 9, and remaining at this level for approximately 5 days, before declining rapidly on days 14 and 15 to reach a low level on the day before oestrus. The progesterone concentration on the day of oestrus was extremely low (0·1 ng./ml.), and was of the same order as that found in the plasma of wethers and anoestrous or ovariectomized ewes. Three ewes, superovulated with pregnant mare serum gonadotrophin, showed marked elevation of peripheral progesterone concentration during the luteal phase of the cycle, the concentration being proportional to the number of corpora lutea formed.