Role of plasma adenosine in breathing responses to hypoxia in fetal sheep.

The importance of plasma adenosine in hypoxic inhibition of breathing movements was determined in chronically catheterized fetal sheep (greater than 0.8 term). Preductal arterial blood for adenosine measurements was withdrawn using a double lumen catheter to mix blood entering the catheter with a solution to stop adenosine metabolism. In 6 fetuses, isocapnic hypoxia (delta PaO2 congruent to -10 Torr) increased the average plasma adenosine concentration from 1.1 +/- 0.2 (SEM) to 2.0 to +/- 0.4 microM. During hypoxia, plasma levels of adenosine were inversely related to preductal arterial O2 content (CaO2) with values ranging between 1.6 and 4.0 microM when CaO2 was less than 3 ml/dl. Hypoxia also significantly reduced the incidence of fetal breathing and rapid eye movements. In other experiments, adenosine (0.36 +/- 0.03 mg/min/kg) was infused for one hour into the inferior vena cava of 5 fetuses. During this infusion, mean plasma concentration of adenosine was 2.8 +/- 0.3 microM, a value about 2.5 times the control average. Adenosine also significantly reduced the incidence of low voltage electrocortical activity, rapid eye movements and breathing activity. We conclude that hypoxic inhibition of fetal breathing most likely arises from an increase in central adenosine production, although during severe O2 deprivation (CaO2 less than 3 ml/dl) blood-borne adenosine could also contribute.     

Transferrin in fetal sheep cerebrospinal fluid and plasma during gestation

1. Transferrin concentrations in fetal sheep CSF and plasma have been estimated between 31 and 125 days gestation and in the adult, using a radial immunodiffusion assay. 2. The plasma concentration was lowest (183 +/- 35 mg/100 ml) in the earliest fetuses examined (31 days). It increased to over 350 mg/100 ml by 35 days; thereafter it was around the adult value (580 mg/100 ml). 3. In CSF the transferrin concentration increased from 43 +/- 10 mg/100 ml at 31 days to a maximum of 163 +/- 14 mg/100 ml at 40 days gestation after which it decreased considerably to 6.1 +/- 0.7 mg/100 ml at 125 days and was even lower in the adult (1.1 +/- 0.2 mg/100 ml). 4. CSF: plasma ratios for transferrin especially when compared with those of other plasma proteins, are not compatible with passive leakage of protein from blood to CSF in the developing brain. The results may be explained by specific transfer of proteins into CSF but synthesis by the choroid plexus or brain has not been excluded.     

Changes in alveolar epithelial cell proportions during fetal and postnatal development in sheep

Basal lung expansion is an important determinant of alveolar epithelial cell (AEC) phenotype in the fetus. Because basal lung expansion increases toward term and is reduced after birth, we hypothesized that these changes would be associated with altered proportions of AECs. AEC proportions were calculated with electron microscopy in fetal and postnatal sheep. Type I AECs increased from 4.8 +/- 1.3% at 91 days to 63.0 +/- 3.6% at 111 days of gestation, remained at this level until term, and decreased to 44.8 +/- 1.8% after birth. Type II AECs increased from 4.3 +/- 1.5% at 111 days to 29.6 +/- 4.1% at 128 days of gestation, remained at this level until term, and then increased to 52.9 +/- 1.5% after birth. Surfactant protein (SP)-A, -B and -C mRNA levels increased with increasing gestational age before birth, but the changes in SP expression after birth were inconsistent. Thus before birth type I AECs predominate, whereas after birth type II AECs predominate, possibly due to the reduction in basal lung expansion associated with the entry of air into the lungs.     

Plasma adenosine and cardiovascular responses to dipyridamole in fetal sheep.

The effects of dipyridamole infusion on fetal arterial plasma adenosine level, [ADO], and the systemic cardiovascular system were studied in 10 fetal sheep at 130-135 days gestational age. Dipyridamole (0.25 mg/kg) was infused into the fetuses intravenously during normoxia and hypoxia. Plasma [ADO] was measured using high-performance liquid chromatography, (HPLC), and fetal heart rate and arterial blood pressure were monitored throughout the study. These studies were performed in the absence and presence of theophylline, an adenosine receptor antagonist. During normoxia (PO2, 23.8 +/- 2.0 Torr), dipyridamole infusion increased fetal plasma [ADO] from 0.82 +/- 0.10 microM to 1.41 +/- 0.16 microM within 1 min (P < 0.01) and fetal heart rate from 157 +/- 6 bpm to 174 +/- 7 bpm (P < 0.01), but did not change mean blood pressure. Fetal plasma [ADO] and fetal heart rate returned to basal levels quickly. Treatment with theophylline did not alter the elevation of plasma [ADO] after dipyridamole infusion, but abolished responses of fetal heart rate to dipyridamole infusion. After 15 min of hypoxia with an average arterial PO2 of 15.4 +/- 1.1 Torr, fetal plasma [ADO] increased to 1.15 +/- 0.14 microM (P < 0.01). Dipyridamole infusion then further raised fetal plasma [ADO] to 1.67 +/- 0.27 microM (P < 0.01). The duration of the increase of fetal plasma [ADO] after dipyridamole infusion was no longer in hypoxia than in normoxia, however there was no significant change in the pattern of transient fetal bradycardia and persistent hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in blood flow distribution during the perinatal period in fetal sheep and lambs.

We have measured total blood flows and blood flows per 100 g tissue to major tissues at 120 and 140 days gestation in fetal sheep and at 3 and 21 days of age in lambs (gestation period = 144 +/- 2 days). Between 120 and 140 days gestation, flow per 100 g tissue increased by 74, 150, and 317% in the renal, intestinal, and hepatic arterial beds, but no further significant change in flow was observed at 3 or 21 days postpartum. Blood flows per 100 g to cerebral hemispheres and cerebellar tissues also increased dramatically during late gestation (142 and 121%, respectively), but declined sharply by 3 days postpartum (73 and 75%, respectively). Brain blood flows at 21 days postpartum remained substantially below late gestational levels. Adrenal blood flows per 100 g more than doubled during late gestation, fell by more than half at birth, and only partially recovered by 21 days of age. Blood flows to carcass tissues did not change in late gestation, fell at birth, then partially recovered. Pre- and post-natal increases in brain blood flows were almost entirely attributable to increased perfusion rather than tissue growth, whereas large perinatal increases in flow to the diaphragm paralleled tissue growth. Tissue growth and increased perfusion per 100 g contributed almost equally to increased blood flows to kidneys postnatally, and to adrenal glands and the gastrointestinal tract prenatally.     

Changes in concentration of adenosine triphosphate and adenosine diphosphate in individual preimplantation sheep embryos.

Concentrations of ATP and ADP were measured in 156 sheep embryos by means of an ultramicrofluorescence assay. Stages of preimplantation development measured included unfertilized oocytes through blastocyst-stage embryos. ATP concentrations remained constant through the 8-cell stage; then ATP decreased significantly (p < 0.025) at the morula stage and remained low through the blastocyst stage. ADP concentrations did not change throughout the embryonic stages measured. Decreased levels of ATP with constant levels of ADP caused the ATP:ADP ratio to decrease significantly (p < 0.025) between the 8-cell and morula stages. We suggest that the increase in glucose uptake by sheep embryos observed at the morula stage of development may be due, in part, to a decrease in the ATP:ADP ratio.     

Progestagen metabolites in fetal sheep plasma: the effect of fetal nephrectomy.

Fetal sheep (100-115 days gestation) were surgically implanted with femoral arterial and venous cannulae and then either sham-operated (control) or bilaterally nephrectomized. Following a 5-day recovery period, fetal blood samples (10 ml/48 h) were taken and the steroid sulphate fraction analysed as trimethylsilyl esters by gas-liquid chromatography (g. l.c.). Three progestagen metabolites were repeatedly detected in plasma samples from control and nephrectomized fetuses and identified by g.l.c.-mass-spectrometric techniques as 5 beta-pregnane-3 beta,20 beta-diol, 5 beta-pregnane-3 beta,20 alpha-diol and 5 beta-pregnane-3 alpha,20 alpha-diol. In three control fetuses the plasma concentration of both 5 beta-pregnane-3 beta,20 beta-diol and -20 alpha-diol showed a steady increase from about 0.5 micrograms/ml at 105 days to about 1.5 and 2-2.5 micrograms/ml, respectively, at 143 days gestation. A study in one fetus indicated that the values then fell precipitously by term (147 days) as plasma cortisol concentrations rose. In contrast, whilst no consistent patterns were seen in their concentration in five nephrectomized fetuses the levels were 2-10 times higher than the control values (0.5-10 micrograms/ml) at all stages. The plasma concentration of 5 beta-pregnane-3 alpha,20 alpha-diol was less perturbed by nephrectomy and only showed a slight increase over control values (0.2-0.5 micrograms/ml). Three sham-operated fetuses which aborted following infection also showed increased plasma concentrations of 5 beta-pregnane-3 beta,20 beta-diol and -20 alpha-diol, similar to the nephrectomized fetuses. It is postulated that high levels of circulating progesterone metabolites may reflect induced increases in adrenal endocrine activity culminating in premature activation of those changes in adrenal function which trigger parturition.     

Changes in plasma volume and haematocrit in intact and splenectomized sheep during feeding.

Changes in jugular haematocrit during daily 2-h feeding periods in trained sheep with and without spleens were compared with changes in the concentration of the plasma tracer radio-iodinated human serum albumin. Jugular haematocrit was increased by 16% in intact sheep and 9% in splenectomized sheep 20 min after they started to eat dry rations. The dilution of tracer in plasma, studied after mixing in the vascular system had been completed, showed four phases. Phase 1 was the rate of removal of tracer in the period before eating began. Phase 2 was the sudden increase in plasma radioactivity that occurred in the first 20 min of eating, indicating a loss of plasma from the circulation. Phase 3 was the decrease in tracer concentration during the remaining feeding period and phase 4 was a post-feeding phase, characterized by a slower rate of decrease of tracer than during phase 3, implying that there was significant recycling of tracer during this phase. The sudden increase in plasma radioactivity, initiated by the onset of feeding, represented a reduction in plasma volume of 10-12%. Minimum plasma volume coincided with peak haematocrit values. The reduced plasma volume accounted for the increased haematocrit in splenectomized sheep, but only accounted for about half of the increase in intact sheep. The residual increase in haematocrit in intact sheep was most likely the result of splenic contraction.     

Thalamic lesions dissociate breathing inhibition by hypoxia and adenosine in fetal sheep

The effects of diencephalic lesions on respiratory responses to intra-arterially infused adenosine (ADO) were determined in chronically catheterized fetal sheep (>0.8 term). These studies were designed to test the hypothesis that the inhibitory effects of ADO on fetal breathing, like those of hypoxia, are mediated by the parafascicular nuclear complex (Pf) of the posteromedial thalamus. ADO inhibited breathing [control (C): 26 +/- 2.6, ADO: 4 +/- 1 min/h] in normal fetuses and in a fetus with a lesion that virtually destroyed the thalamus but left intact most of Pf. Neuronal lesions in the diencephalon, produced by injecting ibotenic acid, abolished the inhibitory effects of ADO on breathing (C: 31 +/- 5.1, ADO: 30 +/- 4.5 min/h) when the lesions encompassed Pf or the sector immediately rostral to Pf that retained the capacity to regulate hypoxic inhibition. Smaller lesions created by the insertion of needles also eliminated the depressant effects of ADO when disruptions were within Pf or a rostral component of the thalamic cortical activating system. It is concluded that 1) a medial thalamic sector is critically involved in ADO-induced apnea and 2) ADO-dependent and ADO-independent mechanisms mediate hypoxic inhibition.     

Cerebral oxygen consumption during asphyxia in fetal sheep

Cerebral blood flow and cerebral arteriovenous oxygen content difference were measured in 17 fetal sheep, and cerebral oxygen uptake was calculated. The measurements were made under control conditions and after profound fetal asphyxia induced of uterine blood flow for up to 90 min. In 14 of the fetal sheep, sequential measurements were made to examine hemodynamic changes and cerebral oxygen consumption at comparable intervals up to 36 min of asphyxia. These fetuses initially had elevated blood pressure and lowered heart rate became hypoxemic, hypercarbic, and acidotic. There was an initial decrease in cerebral oxygen consumption. Sequential measurements, however, showed a relative stability in this decreased oxygenation during 4 to 36 min of asphyxia despite a progressive metabolic acidosis. The cerebral fractional oxygen extraction remained unchanged despite a mean pH of 6.98 at 36 min. The calculated cerebral oxygen uptake during asphyxia in all 17 sheep was grouped according to whether the ascending aortic oxygen content was greater or less than 1.0 mmol/l. In the first group with mean ascending aortic oxygen content of 1.3 mmol/l, blood flow to the brain was increased and cerebral oxygen consumption was 85% of control. In the second group with mean arterial blood oxygen content of 0.8 mmol/l, there was a narrowing of the arteriovenous oxygen content difference, but no further increase in cerebral blood flow. Cerebral oxygen consumption was only 48% of control in this more asphyxiated group. We conclude that the degree of hypoxemia in the second group represents a point where physiologic mechanisms cannot compensate, and may be associated with neuronal damage.     

Thymocyte subpopulations during early fetal development in sheep

Phenotypic analysis of thymocytes during fetal development may identify subpopulations which are either absent or difficult to detect in postnatal thymus. A panel of monoclonal antibodies specific for sheep lymphocyte antigens (SBU-T1, -T4, -T8, -T6) was used to identify thymocyte subpopulations in postnatal and fetal sheep. Thymuses were analyzed by two-color immunofluorescence and flow cytometry or by immunohistology. Two-color immunofluorescent staining of postnatal sheep thymus with anti-SBU-T4 and anti-SBU-T8 revealed four relatively distinct subpopulations with particular localizations: a) SBU-T4-T8-, predominantly outer cortex (12%); b) SBU-T4+T8+, inner cortex (74%); c) SBU-T4+T8-, medulla (10%), and d) SBU-T4-T8+, medulla (4%). One- and two-color immunofluorescent analysis of cells from early fetal thymuses demonstrated the appearance of SBU-T8+ cells well before SBU-T4+ cells. Immunohistologic staining of fetal sheep thymus at various stages of gestation (term = 150 days) revealed that lymphoid cells and MHC class II-positive dendritic cells first appeared at 35 days, at which stage the thymic epithelium was weakly positive for class I MHC antigens but negative for class II MHC antigens. The earliest lymphocyte antigens detectable on fetal sheep thymocytes were SBU-LCA and SBU-T1. By 40 days, the antigens SBU-T6, SBU-T4, and SBU-T8 were detectable on a small number of thymocytes; SBU-T8 preceded SBU-T4, and the number of SBU-T8+ thymocytes always exceeded the number of SBU-T4+ thymocytes throughout early gestation. At 50 days, a thymic medulla appeared and thereafter grew rapidly in size. Immunoperoxidase staining of serial sections of the fetal neck revealed cortical-type thymocytes outside the thymus from 40 days onward, before the appearance of a thymic medulla. However, by 60 days, only medullary-type thymocytes were observed either extrathymically or within the interlobular septa of the thymus, indicating that only thymocytes with a medullary phenotype leave the thymus from this stage of gestation.     

Behavioral activity during prolonged hypoxemia in fetal sheep

Experiments were conducted in unanesthetized, chronically catheterized pregnant sheep to determine the fetal behavioral response to prolonged hypoxemia produced by restricting uterine blood flow. Uterine blood flow was reduced by adjusting a vascular occluder placed around the maternal common internal iliac artery to decrease fetal arterial O2 content from 6.1 +/- 0.3 to 4.1 +/- 0.3 ml/dl for 48 h. Associated with the decrease in fetal O2 content, there was a slight increase in fetal arterial PCO2 and decrease in pH, which were both transient. There was an initial inhibition of both fetal breathing movements and eye movements but no change in the pattern of electrocortical activity. After this initial inhibition there was a return to normal incidence of both fetal breathing movements and eye movements by 16 h of the prolonged hypoxemia. These studies indicate that the chronically catheterized sheep fetus is able to adapt behaviorally to a prolonged decrease in arterial O2 content secondary to the restriction of uterine blood flow.     

Catecholamines in fetal blood during birth in man.

Catecholamines and pH were determined simultaneously in human fetal scalp blood during parturition and in umbilical artery blood at birth. During the course of six normal vaginal deliveries with relatively small changes in blood pH about a ten-fold increase in catecholamine concentration was found. During six deliveries complicated by falls in blood pH to < 7.25 much higher values were often encountered.     

Formation of ovarian follicles during fetal development in sheep

The origin of follicle (i.e., pregranulosa) cells that become the somatic component of primordial follicles is obscure. In addition, information regarding the structural changes that accompany the concomitant regression of ovigerous cords and the appearance of primordial follicles is lacking. In the present study, ovine ovaries collected at frequent time intervals between Day 38 and Day 100 of fetal life were examined by light and electron microscopy. To gain new information regarding the origin of follicular cells, incorporation of 5-bromo-2'-deoxyuridine was used to identify proliferating cells at selected stages of development. Based on the location and identity of proliferating cells, apoptotic cells, and sequential changes in histoarchitecture, we hypothesize 1) that most (i.e., >95%) of the granulosal cells in newly formed primordial follicles originate from the ovarian surface epithelium; 2) that the sequential events leading to follicle formation take place entirely within ovigerous cords, with the first follicles forming at the interface of the cortex and medulla; and 3) that the loss (i.e., >75%) of germ cells, but not of somatic cells, within the ovigerous cords is a means by which each surviving oocyte gains additional pregranulosal cells before follicle formation. Conceptual models detailing the chronology of developmental events involved in the formation of primordial follicles in sheep are discussed.     

Changes in hypoxanthine and lactate during and after hypoxia in the fetal sheep with chronically-implanted vascular catheters

The effect of intra-uterine hypoxia on the hypoxanthine and lactate concentration in fetal sheep with catheters chronically implanted was investigated. Experiments were conducted on five fetuses. Sixty-four blood samples from nine hypoxic and recovery periods were analysed. A significant increase of hypoxanthine and lactate occurred in parallel with the fall of arterial oxygen saturation (SaO2) and arterial oxygen pressure (PaO2) during the first 20 min of hypoxia. The elevations in plasma hypoxanthine and lactate were significantly greater during more severe hypoxia than mild hypoxia, as judged from the amount of low oxygen gas mixture given to the ewe (7 or 9%). There were no difference in PaO2 and only minor difference in SaO2 between the two groups. The increase in lactate over 20 min was the same throughout the one-hour period of hypoxia, while the increase of hypoxanthine was less pronounced at the end of the period. This might be due to the fact that hypoxanthine was cleared from fetal plasma at a fairly rapid rate, half of the excess concentration being eliminated after 25 +/- 21 min compared to 85 +/- 47 min for lactate in six experiments post hypoxia. Linear regression analysis revealed a highly significant correlation between hypoxanthine and SaO2, pH and lactate (P less than 0.001). These three variables explained 77% of the variance of hypoxanthine, when calculated by multiple regression analysis.