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The development of diseases in later life, such as diabetes type II, hypertension and cardiovascular disease, is linked to abnormal intrauterine conditions that reduce birth weight. Obviously, fetal development can be disturbed so profoundly, that fetal programming is changed permanently. We have examined the effects of hypoxia, or more precisely hypoxemia, on the fetal hypothalamic-pituitary-adrenal (HPA) axis and lungs using molecular biology techniques in order to elucidate the underlying mechanisms. Chronically catheterized fetal sheep were subjected to a hypoxemia (48 h) without change in arterial pH or paCO2. Major changes occurred, although the degree of hypoxemia was just moderate. There was a transient increase in the fetal plasma ACTH-concentrations with an upregulation of the cortisol-concentrations, which was more pronounced in the older, hypoxemic fetuses (134-136 days of gestation) than in the younger, hypoxemic animals (126-130 days of gestation; term is 145 days). There was an unique, differential regulation for pro-opiomelanocortin messenger RNA (mRNA), the precursor molecule of e.g. ACTH, in the pars distalis and pars intermedia of the pituitary gland. This finding supported the increased bioactivity besides the increased concentrations for ACTH. Simultaneously, there was an increase in the mRNAs of the ACTH-receptor and of the steroid-synthesizing enzymes in the fetal adrenal gland of the older, hypoxemic fetuses. No changes in the fetal plasma androstenedione-concentrations were observed. Clearly, there was a selective increase of the cortisol-synthesis. Growth and maturation of the fetal lung might also have been affected, because of the increase in surfactant-protein A mRNA in the older, hypoxemic animals and the decrease in the insulin-like growth factor-I and its binding protein-5 mRNA in the younger, hypoxemic fetuses. In summary, even a moderate degree of hypoxemia was shown to affect the different levels of fetal organism profoundly, offering a pathophysiological basis for changes in fetal development.

Hypoxemia represents a major stress for the fetus, and is associated with alterations and adaptations in cardiovascular, metabolic and endocrine responses, which in turn may affect tissue growth and differentiation. To determine the effects of hypoxemia on fetal adrenal activity and growth, we subjected sheep fetuses at days 126-130 and 134-136 (term 145 days) to reduced PaO2 by reducing the maternal fraction of oxygen for 48 h (mean reduction of 6.8 mmHg), without change in arterial pH or PaCO2. This stimulus resulted in similar increases in the plasma immunoreactiveACTH response at both ages. Among adrenal steroids, plasma cortisol (C21Delta4) rose in both groups of animals, but plasma androstenedione (C19Delta4) declined marginally, resulting in a pronounced increase in the cortisol:androstenedione ratio in the plasma that was greater and more sustained in the older fetuses. In the younger fetuses, after 48 h of hypoxemia, there were no significant changes in mRNAs encoding steroidogenic enzymes in the fetal adrenal gland. However, in the older fetuses, hypoxemia resulted in significantly increased levels of mRNAs encoding P450scc, P450C21 and 3beta-hydroxysteroid dehydrogenase, but not for P450C17, in the fetal adrenal gland. Levels of IGF-II mRNA in the fetal adrenal gland fell in both groups of fetuses, and this response was greater at the later gestational age. We conclude that sustained hypoxemia is a potent stimulus which activates adrenal steroidogenesis in the late gestation fetal sheep. The resultant increase in cortisol synthesis is associated with decreased expression of adrenal IGF-II mRNA. We speculate that this relationship might influence patterns of fetal organ growth and differentiative function in response to fetal stress such as hypoxemia.

Development of the fetal adrenal gland is crucial not only for maturation of several fetal organ systems and the initiation of parturition, but also for the development of the fetal response to stress. The enkephalin-related peptides are present in the chromaffin cells of the fetal adrenal medulla and are secreted in response to stress and with sympathetic stimulation. However, changes in expression of preproenkephalin (PENK) with gestation and in response to stress have not been studied in detail. Therefore we examined the developmental pattern of PENK gene expression in the adrenal gland of fetal and newborn lambs, and of adult sheep. We also determined whether levels of PENK mRNA in the fetal adrenal gland changed in response to exogenous glucocorticoids in late gestation, or in response to hypoxemia. Adrenal glands were removed from fetal sheep, lambs and adult sheep at different stages of development for measurement of PENK mRNA. Cortisol was infused (5 micrograms/min) for 12, 24 or 96 h beginning on day 124-129 of gestation. Moderate hypoxemia was induced for 48 h beginning on day 126-130, or at day 134-136 of gestation, by lowering the maternal fractional inspired oxygen. At the end of the treatment periods, the ewes and fetuses were euthanized. Adrenal PENK mRNA were measured by Northern blot analysis. PENK mRNA levels in fetal adrenals were significantly higher (P < 0.05) on days 140-141 of gestation than earlier in pregnancy, and then decreased significantly with the onset of parturition (days 142-146). After cortisol infusion to the fetus for 96 h there was a significant reduction in adrenal PENK mRNA levels. Hypoxemia resulted in a significant increase in PENK mRNA levels in fetuses at day 126-130 of gestation, but not at the later time in pregnancy when endogenous plasma cortisol concentrations were higher. We conclude that there is a decrease in levels of PENK mRNA in the fetal adrenal gland before parturition at the time of the endogenous prepartum rise in plasma cortisol. Hypoxemia led to an elevation of PENK mRNA levels in fetuses at less than 130 days, but after that time, when the basal and stimulated cortisol responses had risen, there was no significant effect of hypoxemia on PENK mRNA. Cortisol infusion to the fetus at this stage of pregnancy resulted in a decrease in adrenal PENK mRNA levels. We suggest that cortisol may play an important role in the regulation of fetal adrenal PENK mRNA levels and enkephalin synthesis by the adrenal gland of the fetal sheep.

POMC messenger RNA (mRNA) is synthesized and translated to form ACTH in the pars distalis and ACTH-related peptides in the pars intermedia of the pituitary in fetuses of species such as sheep. In the fetal sheep, maturation of hypothalamo-pituitary-adrenal (HPA) function occurs during late pregnancy accompanied by increased levels of POMC mRNA in the pars distalis, and contributes to the stimulus for birth. Activation of fetal HPA function also occurs with short term hypoxemia. However, the effects of more prolonged fetal hypoxemia, which may predispose to premature parturition, on POMC mRNA levels in the pars distalis and pars intermedia are not known. We studied the effects of sustained hypoxemia (48 h) in the absence of acidosis on fetal HPA responses at two times in late gestation, days 126-130 and days 134-136 (term = approximately 145 days). Hypoxemia was induced by lowering the fraction of oxygen in the maternal inspired gas mixture. Fetal arterial oxygen tension fell by 6-8 mm Hg, without a change in fetal arterial pH or carbon dioxide tension. Pituitary POMC mRNA was localized and quantified by in situ hybridization. At both gestational ages, hypoxemia caused a transient elevation in fetal plasma ACTH, with a maximum response at +2 h, and a sustained elevation in circulating cortisol. Cortisol responses were greater in the older fetuses. Changes in plasma cortisol were not associated with alterations in the plasma corticosteroid-binding capacity or in levels of hepatic corticosteroid-binding globulin mRNA. Pituitary POMC mRNA showed a regional distribution in the pars distalis, and mean levels increased with gestational age. After 48 h of hypoxemia, POMC mRNA levels had increased in the pars distalis. The regional distribution was unaffected by the hypoxemic insult. In contrast, POMC mRNA levels in the pars intermedia decreased after hypoxemia, and the reduction was greater in the older fetuses. We conclude that in response to prolonged hypoxemia, there is differential regulation of POMC mRNA levels in the pars distalis and pars intermedia of the pituitary in fetal sheep. The magnitude of these responses also changes with gestational age. The increase in POMC mRNA levels in the pars distalis at 48 h of hypoxemia, despite a rise in plasma cortisol and without a change in corticosteroid-binding globulin, suggests altered regulation of HPA function at this time.

In a previous study on acute asphyxia in unanesthetized fetal sheep near term we showed that reduced oxygen delivery to peripheral organs reduces total oxygen consumption, suggesting that oxygen itself may be a determinant of oxygen consumption (Jensen, Hohmann & Künzel, 1987). To test this hypothesis we developed an in vitro perfusion model, which enabled us to measure the oxygen consumption of fetal skeletal muscle cells in monolayer culture in a control period (at approximately 145 mmHg) and during various degrees of hypoxia (6-140 mmHg). In 57 experiments on 57 cultures the mean oxygen consumption at a mean 'entry PO2' of 145.3 +/- 10.4 mmHg was 10.3 +/- 9.3 (SD).10(-6) microliters O2 per h per skeletal muscle cell. These measurements were made after an average of 4.2 +/- 2.3 transfers of the cells and at a cell density of 2.0 +/- 1.2.10(5) cells per cm2. In 54 of these experiments hypoxia was induced. There was a close positive correlation between the PO2 of the perfusate entering the Petridish ('entry PO2') and the change of the oxygen consumption of the cells (y = 5.17 - 0.54x + 0.03x2 - 0.00016x3, r = 0.97, p less than 0.0001). When oxygen tension fell, there was a concomitant fall in cellular oxygen consumption. We conclude that oxygen is a determinant of cellular oxygen consumption. Thus, hypoxia may reduce oxygen consumption of skeletal muscle cells, and oxygen may be preserved to maintain oxidative metabolism in central fetal organs.(ABSTRACT TRUNCATED AT 250 WORDS)