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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.

Prostaglandin (PG) F(2alpha) secreted from the uterus is the luteolysin of the estrous cycle and is also believed to be responsible for luteolysis in the pregnant doe at term. We have reported that basal progesterone concentrations decrease before basal PGF(2alpha) concentrations increase, which is inconsistent with this view. In this study we investigated whether luteolysis is associated with increased frequency or amplitude of pulsatile PGF(2alpha) secretion in does over the last 2 wk of gestation. Progesterone concentrations decreased approximately 1 wk before parturition. There was no accompanying increase in PGF(2alpha) concentrations or pulse frequency, and those pulses that were observed were of lesser amplitude and duration than those that have been associated with luteolysis in cycling ewes. A small increase in PGF(2alpha) pulse frequency was identified during the 3 days before parturition, but this was not associated with any change in progesterone concentrations. The biological significance of these small changes in PGF(2alpha) pulse frequency is obscure, although the high concentration of this eicosanoid at labor may have been related to the final, precipitous decline in plasma progesterone concentrations. These findings do not support the notion that PGF(2alpha) is the principal luteolysin in the pregnant doe at term.

We investigated the temporal relationship of fetal cortisol secretion to circulating concentrations of fetal ACTH1-39 and its high-molecular weight precursors in goats. We also measured the concentrations of progesterone, estradiol-17beta estrone sulfate, prostaglandin (PG) E, PGF2alpha, and PGF2alpha metabolite (PGFM) in maternal arterial plasma over the last month of gestation. Prostaglandin concentrations were also measured in utero-ovarian venous plasma. There was a positive association between ACTH1-39 in fetal plasma and the prepartum surge in fetal cortisol that commenced 8 days before labor. The fetal cortisol surge was followed by a simultaneous decrease in maternal progesterone and an increase in plasma estrogens commencing 3-4 days before labor. No change in basal prostaglandin concentration occurred before this time. There was a positive veno-arterial difference of PGE and PGF2alpha across the uterine vascular bed, confirming the uteroplacental unit as a major source of these eicosanoids in the plasma of the pregnant doe. We conclude that the fetal signal for parturition precedes luteolysis by some 5 days and find no evidence of changes in the basal concentrations of PGE and PGF2alpha in maternal plasma at the time of luteolysis.

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.

Arterial blood gases, pH and haemoglobin concentrations were monitored for 20 minutes before, during and for 120 minutes after 60 seconds of electroimmobilisation (E-IM) at current strengths of 0 mA (control), 40 mA and 60 mA in 17 Merino ewes (36.3 +/- 1.0 kg) previously prepared with unilateral carotid artery loops. E-IM elicited whole body rigidity. During E-IM, breathing was stopped for 50 +/- 3 seconds (40 mA) and 56 +/- 2 seconds (60 mA). Arterial PO2 decreased to 43.2 +/- 2.4 mmHg (5.68 +/- 0.32 kPa) (40 mA) and 36.4 +/- 1.8 mmHg (60 mA) while arterial PCO2 rose to 59.7 +/- 1.9 mmHg (40 mA) and 69.8 +/- 2.0 mmHg (60 mA). There was a significant fall in arterial pH to 7.272 +/- 0.014 (40 mA) and 7.233 +/- 0.011 (60 mA) during E-IM and arterial haemoglobin increased by 34 +/- 3 per cent and 35 +/- 3 per cent at 40 mA and 60 mA, respectively. All the arterial blood gas and pH changes were significantly greater (P less than 0.05) during E-IM at 60 mA than at 40 mA. Multiple regression analysis indicated that the decrease in arterial PO2 during E-IM was directly related to the latency to breathe while the changes in arterial PCO2 and pH during E-IM were not.2+off

The effects of exogenous oxytocin (OT) and estradiol-17 beta (E2) on plasma concentrations of prostaglandin (PG) E2 and 13, 14-dihydro-15-keto-PGF2 alpha (PGFM) were investigated on Day 14-15 (NP) of the estrous cycle and Days 14-16 (PI) and 21-25 (EP) of pregnancy in the ewe. Basal concentrations of PGFM were significantly elevated in utero-ovarian venous (UOV) plasma on Day 14 of pregnancy (4.05 +/- 0.81 nM, mean +/- SEM) compared to that observed on Day 14 of the cycle or Days 21-25 of pregnancy (2.29 +/- 1.3 nM and 1.06 +/- 0.56 nM, respectively). PGFM release increased significantly following intera-arterial bolus injections of 50, 500, and 5000 mU OT at 2-h intervals in all experimental groups. There was no significant difference in area and peak height of the PGFM response between the 3 groups studied. The time to peak PGFM response was, however, significantly longer in the PI group. No significant changes in concentration of PGFM were observed in any experimental group following 1-h infusions of E2 at 5, 50, and 500 pmol/min. Long-term (15-18 h) infusion of E2 at 83 pmol/min increased the peak height of the OT-induced PGFM response at both stages of gestation studied. PGE2 concentrations in UOV plasma were less than 0.05 nM in all samples studied. These results demonstrate that PG release can be induced in response to OT during the period in which ovine trophoblastic protein-1 (oTP-1) is released by the conceptus. During pregnancy, oTP-1 does not appear to inhibit the E2 induction of uterine OT receptors.

A polarographic oxygen electrode technique was used to quantify the activity of prostaglandin G/H synthase present in microsomes prepared from cotyledons of ewes at 74 to 147 days of gestation (n = 25). Microsomal oxygen consumption was stimulated by arachidonic acid and suppressed significantly (p less than 0.0001) by indomethacin, a specific inhibitor of prostaglandin G/H synthase. A residual indomethacin-incentive oxygen consumption was identified but the magnitude of this component did not change during gestation. During gestation prostaglandin G/H synthase (PGHS) activity, present in ovine cotyledons, increased 22-fold, from 0.6 +/- 0.19 at 70-80 days (n = 3) to 13.22 +/- 2.08 nmol O2/min/mg protein at greater than 145 days of gestation (n = 8; p less than 0.001). This gestational increase in the PGHS activity present in ovine cotyledons was not observed when fetal hypophysectomy was performed at 70-80 days of gestation. The data obtained in this study are consistent with the hypothesis that cotyledonary PGHS activity is increased during the second half of gestation and that this increase may be dependent upon fetal pituitary factors.

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 ultrastructure of the corpus luteum of the sheep has been examined throughout the estrous cycle. Small, densely staining granules 0.2 im in diameter, are observed within the luteal cell at Day 2 of the cycle; they increase in number as the cycle progresses and at Day 6 the first signs of secretion are noticed. Maximum secretion of granules takes place at Days 10 and 11, and this is followed by a progressive decline in secretory activity from Day 12 to Day 15 of the cycle. These clear-cut changes in granule formation and secretion therefore reflect the established pattern of progesterone secretion by the ovary during the estrous cycle in the sheep. Fractions were obtained from corpora lutea of sheep by homogenization and differential centrifugation. A correlation was obtained between the presence of densely staining granules (approx 0.2 um diam) and the progesterone content of each fraction. Many types of nonsteroidogenic endocrine cells are known to have a secretory pathway whereby hormones are packaged in the Colgi region and then discharged from the cells via granules; the present work provides the first indication of a steroid hormone, viz, progesterone, being secreted in a similar way.

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.

Concentrations of ovine placental lactogen (oPL) were measured in the peripheral plasma of six ewes after the removal of fetuses (in four ewes) or after the intrauterine death of one of twin fetuses (in two ewes). Concentrations of oPL decreased after fetectomy or intrauterine fetal death, indicating that a living fetus is essential to the sustained secretion of oPL.

It is uncertain whether ovarian bloodflow in ewes remains constant or varies with the stage ofthe oestrous cycle. In an experiment in whichminimal estimates' of the ovarian steroid secretion rates were obtained, Short, McDonald & Rowson (1963) cannulated the "major vein draining the corpus luteum or ripe Graafian follicle". The bloodflow rates obtained were lower in oestrous than in di-oestrous ewes. Moore, Brown & Smythe (1965) using a similar technique did not find that the rates varied cyclically. In the present study, seventeen Merino and five Corriedale ewes were used. They were 5 to 7 years old and had regularly exhibited oestrus at intervals of 17 days. Anaesthesia was induced and maintained with sodium pentobarbitone except on nine occasions when anaesthesia was maintained with fluothane. The abdomen was opened by a mid-ventral incision and 12,500 i.u. of heparin were given intravenously. A polyvinyl catheter was passed into each utero- ovarian vein through an incision in a branch of the corresponding middle uterine vein. Each utero-ovarian vein was ligated below the end of the catheter. Tributaries of this vein, other than the ovarian veins, were also ligated so that only ovarian blood entered the catheter. Each catheter (length 9 cm, internal diameter 2\m=.\0mm) was connected with 3\m=.\2-mminternal diameter tubing to form a low-resistance collection system. Timed collections of blood, approxi- mately 80 ml from each ovary, were made simultaneously. The ligatures and catheters were then removed and the incised veins tied off. The abdomen was closed and the animal allowed to recover. Collections were made on 2 successive days in twenty of the ewes. In twelve of these, the ovaries were removed and weighed after the second collection period. In individual ewes during the luteal phase, similar bloodflow rates were obtained at the two collections. Ovarian bloodflow during consecutive periods of sodium pentobarbitone-maintained and fluothane-maintained anaesthesia was similar in each of three ewes examined. Consequently the results from all animals were pooled. For Days 0, 1, 2, 15 and 16 of the oestrous cycle (Text-fig. 1), the mean bloodflow ( + S.E.) was 2-91+0-25 ml/min in ovaries with pre-ovulatory follicles or recent ovulation points and 3-05 + 0-32 ml/min in the contralateral 'inactive' ovaries. The rates during Days 3 to 14, inclusive, of 7-80 + 0-48 ml/min in ovaries with corpora lutea and 6-59 + 0-42 ml/min in those without corpora

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.

We have compared six different solutions commonly used for the extraction of peptides to optimize the extraction of Epidermal Growth Factor (EGF) from mouse submandibular glands. The yield of EGF was always higher in neutral pH extractions than in acidic ones. However, the EGF extracted at neutral pH was only poorly adsorbed on to octadecylsilyl silica, whereas the EGF extracted at acidic pH was be easily adsorbed. Subsequent purification of the EGF extracts by two-stage reversed-phase High Performance Liquid Chromatography yielded highly purified EGF. Identical chromatograms were obtained from each of the different extracts. Preparative scale quantities of EGF could be purified rapidly and reproducibly at high efficiency from acidic extracts.

The effects of nutrient availability on fetal plasma prostaglandin E (PGE) concentrations, on fetal breathing movements and electromyographic (EMG) activities of fetal nuchal and forelimb muscles were investigated in pregnant ewes by varying dietary intake and by manipulation of fetal plasma glucose concentration. The incidence of fetal breathing movements (06.00-10.00 h) decreased with increasing gestational age while fetal arterial concentrations of plasma PGE increased significantly over the same period of gestation. Maternal fasting for 48 h reduced the incidence of fetal breathing movements and the amount of nuchal EMG activity (06.00-10.00 h) in animals older than 130 days but had no effect earlier in gestation. No changes in forelimb EMG activity were observed during fasting at any gestational age. Plasma PGE levels increased significantly during fasts begun both before and after 130 days of gestation. When data from fed and fasted states were combined for all fetuses, irrespective of gestational age, there was a significant inverse correlation between fetal breathing movements incidence and plasma PGE concentration in utero. This relationship was even more pronounced when the fetuses were considered individually. Insulin infusions induced hypoglycaemia, an increase in fetal plasma PGE concentration and a significant reduction in the incidence of fetal breathing movements at all ages. Glucose infusions of fetal breathing movements only after 130 days and had no effect on plasma PGE levels in utero at any gestational age. Neither insulin nor glucose infusions altered the EMG activities of the nuchal and forelimb muscles. The results show that glucose availability is an important factor in determining the incidence of fetal breathing movements in utero and indicate that nutritionally induced changes in fetal breathing movements are mediated in part by PGE. They also suggest that PGE is a physiological regulator of fetal breathing movements in the sheep during late gestation.

The mechanisms involved in the control of oxytocin and progesterone secretion by the ovine corpus luteum have been investigated in vitro using luteal slice incubations. Oxytocin and progesterone were secreted at constant rates from luteal slices for 2 h of incubation (366 +/- 60 pg X mg X h and 18.9 +/- 0.18 ng X mg X h, respectively). Secretion of progesterone, but not of oxytocin, was significantly (p less than 0.02) stimulated in the presence of ovine luteinizing hormone. Incubation of luteal slices in medium containing 100 mM potassium, however, resulted in increased secretion of oxytocin and, to a lesser extent, of progesterone (294 +/- 59% and 142 +/- 15%, respectively, p less than 0.05). Basal oxytocin secretion was reduced during incubation in calcium-free medium, compared to secretion in the presence of calcium (70 +/- 15 and 175 +/- 25 pg X mg X 20 min, respectively, p less than 0.01), whereas progesterone secretion was not altered in the absence of calcium. Secretion of both hormones by luteal slices was stimulated by the addition of the calcium ionophore A23187 (p less than 0.05). Addition of prostaglandin F2 alpha (2.8 microM) had no effect on secretion of either oxytocin or progesterone. We have demonstrated that oxytocin and progesterone can be stimulated, independently, from corpus luteum slices incubated in vitro. The pattern of release is consistent with the proposal that oxytocin, but not progesterone, is associated with and actively released from luteal secretory granules. Our results also indicated that prostaglandin F2 alpha does not directly stimulate release of oxytocin or progesterone from luteal cells in vitro.

The possible rôle of the fetal hypothalamic-pituitary axis in regulating the secretion of ovine placental lactogen (oPL) was investigated in chronically-catheterised ewes and fetuses in late pregnancy. Intravascular administration of agents to fetuses that significantly increased fetal prolactin concentrations (chlorpromazine 6.25 mg;thyrotrophin releasing hormone, 10 micrograms), significantly reduced fetal prolactin concentrations (bromocriptine, 0.033 mg/h), or significantly reduced fetal growth hormone (GH) concentrations (somatostatin, 2.5 micrograms/min), had no effect on maternal or fetal oPL concentrations. Mean fetal levels of prolactin or GH in late gestation could not be correlated with oPL concentrations, although fetal hypophysectomy prevented the normal prepartum fall in oPL concentrations.

We have measured the changes in fetal and maternal plasma concentrations of cortisol in relation to blood gases and percent oxygen saturation during 2- and 4-h episodes of reversibly reduced uterine blood flow in sheep between 120 days gestation and term. During that period of reduced uterine blood flow there was a significant decrease in fetal arterial percent oxygen saturation (SaO2), PO2 and pH. Fetal SaO2 decreased from 59.5 +/- 3.2% to 31.8% +/- 2.8% by 15 min, 32.9 +/- 2.9% by 60 min, and 33.5 +/- 2.9% by 120 min. Fetal PO2 decreased from 3.2 +/- 0.1 KPa to 2.0 +/- 0.2 KPa by 15 min, 2.2 +/- 0.2 KPa by 60 min and 2.3 +/- 0.1 KPa by 120 min. Fetal pH decreased from 7.36 +/- 0.01 to 7.30 +/- 0.03 by 15 min, 7.27 +/- 0.02 by 60 min and 7.25 +/- 0.03 by 120 min. During the period of reduced uterine blood flow, fetal plasma concentrations of cortisol increased from 37.1 +/- 10.8 nmol/l to 53.3 +/- 9.2 nmol/l by 15 min, 49.2 +/- 11.4 nmol/l by 60 min and 43.3 +/- 9.0 nmol/l by 120 min. The greatest percentage increase in fetal plasma concentrations of cortisol occurred in fetuses of 126-139 days gestation. There was no significant change in maternal blood gases, SaO2 or plasma concentrations of cortisol. These experiments demonstrate that there is a significant increase in fetal plasma concentrations of cortisol in response to reductions in uterine blood flow from as early as 120 days gestation.

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.

Haemoglobin synthesis has been studied in fetal sheep. The switch from gamma to beta chain synthesis occurs rapidly between 130 and 150 days. Fetal hypophysectomy delays the rate of switching but the timing of the onset of the switch is not under pituitary control.

Experimental intrauterine growth retardation was studied in sheep. Endometrial caruncles (anlagen of maternal cotyledon) were removed before pregnancy and at a second operation, catheters were implanted into the ewe and fetus at 105-135 days of pregnancy. Three groups of fetuses were defined: low birthweight-for-dates (small-caruncle), normal birthweight-for-dates (normal-sized-caruncle) from ewes which had endometrial caruncles removed and the controls. The mean placental weights in these groups were 139 plus or minus 5 g, 283 plus or minus 46 g, 334 plus or minus 22 g respectively. The brains, kidneys and adrenals of the small-caruncle-fetuses were significantly greater in proportion to body weight than in the controls and the appearance of ossification centres was delayed. Arterial oxygen tension was lower and packed cell volume higher in the small-caruncle-fetuses (PaO2 15 plus or minus 0.6 mmHg; packed cell volume 37.3 plus or minus 1.6%) and normal sized caruncles (PaO2 20.7 plus or minus 1.2 mmHg; packed cell volume 35.2 plus or minus 0.7%) than in the controls (PaO2 23.2 plus or minus 0.7 mmHg; packed cell volume 29.8 plus or minus 0.7%). Plasma concentrations of glucose (0.65 plus or minus 0.12 micromol/ml), lactate (0.9 plus or minus 0.1 micromol/ml) and pyruvate (0.08 plus or minus 0.025 micromol/ml) were lower in small-caruncle fetuses than in the control fetuses (glucose 1.05 plus or minus 0.06 micromol/ml, lactate 1.83 plus or minus 0.7 micromol/ml, pyruvate 0.21 plus or minus 0.06 micromol/ml). The corresponding values for the normal-sized-caruncle fetuses were glucose 0.71 plus or minus 0.12, lactate 1.18 plus or minus 0.7 and pyruvate 0.12 plus or minus 0.03 micromol/ml. The plasma concentration of alanine in the small-caruncle-fetuses (0.25 plus or minus 0.09 micromol/ml) was higher than in the normal-sized-caruncle (0.073 plus or minus 0.009 micromol/ml) or control fetuses (0.12 plus or minus 0.013 micromol/ml). The results indicate that fetal growth retardation due to restriction of placental growth after removal of endometrial caruncles is associated with chronic hypoxaemia, polycythaemia and hypoglycaemia. The restriction of nutrient supply probably accounts for the altered pattern of fetal growth but the relative importance of the changes observed remains uncertain.

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.