0013.7227/92/1305-2571$3.00/O
Endocrinology Copyright 0 1992 by The Endocrine
Vol. 130, No. 5 Printed in U.S.A.
Society
Dissociation of Pulsatile Cortisol and Adrenocorticotropin Secretion in Fetal Late Gestation* EDE AND
MARIE STEVEN
APOSTOLAKIS, M. YELLON
LAWRENCE
D. LONGO,
JOHANNES
Sheep during D. VELDHUIS,
Division of Perinatal Biology, Departments of Physiology, Pediatrics, and Gynecology and Obstetrics, Loma Linda University, School of Medicine, Loma Linda, California 92350; and Division of Endocrinology and Metabolism, Department of Internal Medicine (J.D. V.), University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
ABSTRACT.
In the fetal sheep, plasma cortisol concentrations gradually increase in the last weeks of gestation and abruptly rise during the final 48-72 h preceding birth. To determine if these changes in mean circulating cortisol concentrations result from increased pulsatile secretion and are driven by changes in ACTH pulses, blood samples from five chronically catheterized fetuses were collected every 5 min for 2 h at 133 days gestation and every 4 days thereafter until delivery at 146 + 2 days. Volume was replaced after each blood sample and erythrocytes were returned every 20 min. Plasma cortisol and ACTH secretion were pulsatile in fetuses at all ages. Cortisol pulse frequency increased significantly with gestation from a mean of 2.2 pulses/2 h at 133 days to 4.8 pulses/2 h at 146 days. The interpulse interval (mean + SE) decreased between 133 and 146 days from 54 + 11 min to 23 + 3 min, respectively. Cortisol pulse amplitude increased significantly from 10 + 2 rig/ml at
I
N THE fetal sheep, cortisol in circulation gradually increases over the last third of gestation to abruptly rise during the 72 h preceding birth (1, 2). This surge in fetal cortisol precedes the endocrine cascade which initiates parturition (3, 4). In parallel with changes in cortisol in circulation, fetal plasma ACTH also increases, but less dramatically (5-8). These findings are based on daily and/or less frequent blood sampling protocols. To define the temporal relationship between the secretion of the trophic hormone ACTH and the response of its target organ, the adrenal cortex, more frequent assessment of concentrations in circulation is required. PulsaReceived September 23, 1991. Address all correspondence and requests for reprints to: Steven M. Yellon, Ph.D., Division of Perinatal Biology, Loma Linda University, School of Medicine, Loma Linda, California 92350. * This work was supported by grants from the US Public Health Service (HD-03817 to L. D. L.) and the Center for Nursing Research (NRSA-06042 to E. M. A.), and the Pediatrics Department Research Fund, Loma Linda University School of Medicine. Preliminary work was presented at the 23rd Annual Meeting of the Society for Study of Reproduction, July 17-21, 1990, Knoxville, Tennessee (No. 413).
133 days to 44 + 13 rig/ml at 146 days. In contrast to cortisol, ACTH pulse frequency (3 + 0.6 pulses/2 h) and amplitude (21 + 3 pg/ml) were similar at 133 days and 146 days. The coincidence of cortisol and ACTH pulses did not change between 133 and 146 days. Furthermore, the number of coincident pulses failed to exceed random associations (hypergeometric probability analysis) and could have occurred by chance alone (P values ranged from 0.11-0.63). A point by point comparison of cortisol and ACTH concentrations in fetal circulation indicate that only 36% of the variance in cortisol concentrations could be explained by variance in ACTH (cross-correlation analysis). These data suggest that fetal cortisol and ACTH secretion are pulsatile and that, as gestation advances, increases in constitutive cortisol pulse amplitude and frequency may not be predominantly driven by pulsatile changes in ACTH in the ovine fetal circulation near term. (Endocrinology 130: 2571-2578, 1992)
tile ACTH release occurs in adults of various species, including rats (9), dogs (lo), and sheep (ll-13), as well as from human fetal pituitaries in vitro (14). In the fetal sheep, evidence suggests that concentrations of cortisol and ACTH in the circulation vary within short time intervals (15-17). The goal of the present study was to determine whether plasma cortisol and ACTH secretion is pulsatile in fetal sheep. Since increased adrenal responsiveness to exogenous ACTH stimulation occurs in fetal sheep during late gestation (18, 19) and tissue responsiveness to a trophic hormone can be controlled by patterns of pulsatile secretion (20), we also tested whether increasing fetal cortisol during late gestation was temporally associated with enhanced pulsatile ACTH secretion.
Materials
and Methods
General
Studieswere performed in five time-dated pregnant singleton ewesof mixed Western breed (Nebeker Farms, Santa Monica, 2571
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FETAL OVINE PULSATILE
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CA). Ewes experienced 10 h of light each day (lights on 0700 h, PST) in a room with constant humidity and temperature. A dim red light ( 0.05, Kolmogorov-Smirnov test). In summary, less than one third of the variance in cortisol concentrations were correlated to changes in ACTH concentrations, a relationship that is reminiscent of the one third of ACTH pulses that were coincident with peak pulses of cortisol in fetal circulation (Fig. 3), and which was not statistically distinguishable from random occurrence. Discussion
The results provide the first in uiuo evidence that endogenous basal secretion of cortisol and ACTH in fetal sheep is pulsatile during the last 2-3 weeks of gestation. The data indicate that the rise in mean plasma cortisol concentration in the fetus with gestation can be attributed to an increase in both frequency and amplitude of cortisol pulses in circulation, assuming no change in hormone half-life. The results also suggest that this rise in fetal cortisol is not entirely driven by fetal ACTH. Although ACTH secretion is also pulsatile in the ovine fetus, there was little evidence for a change in either pulse frequency or amplitude near term. These data, in TABLE
1. Range
and median
cross-correlation
(r) values
between
plasma
HORMONE
age
-10
cortisol
-5
Range
Median
133
0.01-0.69”
137
0.01-0.56
0.28 0.17
142 146
-0.08-0.76 -0.16-0.23
a P < 0.05 indicates
a nonrandom
0.27 0.012
distribution
Range 0.27-0.80" 0.15-0.73" 0.09-0.78" 0.01-0.21
between
and ACTH
concentration
Range
0.44 0.21 0.48 0.20
plasma
cortisol
in fetal sheep
lag (min) +10
+5
0
Median
2575
conjunction with the finding that a majority of cortisol pulses near delivery are not associated with pulsatile ACTH secretion, suggest that ACTH may not be the principal trophic drive of constitutive pulsatile cortisol secretion in the fetal sheep during the days preceding parturition. An alternative to this interpretation is that a pulse of ACTH provokes a volley of pulses or sustained release of cortisol. Cortisol secretion is enhanced in sheep during the weeks preceding delivery as the fetal adrenal matures (6). In previous studies of fetal sheep older than 135 days gestation, increases in ACTH concentration of 80-100 pg/ml elevated plasma cortisol levels for less than 15 min while ACTH concentrations of more than 500 pg/ml stimulated high cortisol levels for 30 min (17, 28). In the present study of fetuses at 146 days, mean ACTH concentrations and nadir were 60 f 12 and 53 + 6 pg/ml, respectively. Further, on only one occasion during a pulse study did maximal ACTH levels exceed 81 pg/ml. Thus, it seems unlikely in the present study of constitutive secretion that peak episodes in ACTH secretion could account for a pulse volley or sustained release of cortisol. It should be recognized that data derived from blood samples collected at an interval of 5 min over 2 h has the inherent potential to underestimate high frequencylow amplitude pulsatile hormone secretion. We estimate that up to 4 cortisol pulses/2 h and 8 ACTH pulses/2 h could be clearly resolved (maximum amplitude of 15 ng/ ml and 5 pg/ml, respectively) without an upward drift in mean nadir concentration. These estimates are empirically based on the half-life of the respective hormones (29, 30) and the absence of change in nadir hormone levels during the 2-h pulse study. In the present study, cortisol pulse amplitudes were rarely lower than those stated above. In addition, a rise in mean cortisol nadir concentration with gestation was associated with increased cortisol pulse frequency and amplitude. By contrast, the ACTH nadir concentration failed to change with gestational age, and throughout the study, pulse frequency was well below the estimated 8 pulses per 2 h, the upper limit of detection. In addition, ACTH pulse amplitude was comparable to that reported by Engler et al. (12) in adult sheep using a 2-min sampling interval.
Time Gestation (days)
SECRETION
Median
Range
Median
0.01-0.84 0.04-0.70
0.34 0.29
0.25-0.70" 0.08-0.43"
0.54 0.25
0.31-0.75" 0.15-0.35"
0.59 0.36
0.39-0.68" 0.05-0.40
0.49 0.25
and ACTH
concentrations
Range
Median 0.25
0.37-0.70" 0.05-0.62 0.12-0.53"
0.21 0.34 0.08
0.08-0.27
by the Kolmogorov-Smirnov
test (25).
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FETAL
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Moreover, even with a 2-min sampling interval, pulse amplitude may be underestimated. Thus, there is little evidence to suggest that a high frequency-low amplitude pattern of ACTH secretion remained undetected or is biologically relevant for regulating cortisol pulses in the ovine fetus (30a). The potential for fetal stress was another concern in this study. The intensive blood withdrawal protocols required to demonstrate spontaneous variations in constitutive ACTH and cortisol in circulation could be confounded by stress-related secretion. A decrease in total circulating blood volume greater than 7% has been reported to initiate hypothalamic release of arginine vasopressin which, in turn, stimulates release of ACTH and cortisol (31, 32). In the present study, we withdrew less than 3% of circulating volume in total. Moreover, blood volume was immediately replaced and erythrocytes returned every 20 min, in effect virtually no loss in total circulating blood volume. The conclusion that the 5-min blood withdrawal protocol did not stress the fetus or stimulate ACTH release is further supported by the lack of correlation between pulsatile hormone secretion and either the sampling frequency or erythrocyte replacement. In addition, the amplitude of ACTH and cortisol pulses and means of each 2-h pulse study at all gestational ages in the present project were within limits of previous reports (6-8, 15), and typically well below those observed in fetal sheep with mild hypovolemia (31, 32) or after receiving exogenous AVP (15, 33). Finally, arterial blood gases remained stable and within normal range during each pulse study. The crucial question remains what drives the gestational rise in fetal cortisol secretion. There is little to suggest that fetal ACTH is the principal modulator of constitutive cortisol secretion in the present study, because one third or fewer of cortisol pulses were coincident with ACTH pulses during late gestation and a maximum of 36% of the variance in cortisol concentration could be explained by ACTH levels (square of the median r value in Table 1). By contrast, in adult sheep, at least 70% of the recognized cortisol pulses were associated with pulses of ACTH in circulation (12). Others have proposed that the ovine fetal adrenal becomes more sensitive to ACTH as term approaches (6, 34-36). By definition, if adrenal sensitivity to ACTH increased with gestation, an ACTH pulse of similar amplitude and frequency at 133 and 145 days should induce increased cortisol secretion at 146 days. In the present study, amplitude of cortisol pulses increased at 146 days compared with 133 days. Further, coincidence of conjoint ACTH and cortisol pulses were increased while the number of isolated ACTH pulses (those not associated with cortisol pulses) decreased at 146 days compared with 133 days. Thus, the data cannot exclude the possibility that adrenal sensitivity to ACTH
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Endo. Voll30.
1992 No 5
increases with fetal maturation. However, the number of cortisol pulses not associated with ACTH secretion dramatically increased and the distribution of cortisol pulses was more random relative to an ACTH pulse at lag times of +5 min, +lO min (table l), and +15 min (data not shown) near term. Collectively, the data raise the possibility that a trophic factor other than immunoreactive ACTH may drive the marked rise in circulating fetal cortisol at the end of gestation. Removal of an inhibitor of fetal adrenal steroidogenesis may also account for the gestational rise in fetal cortisol. High molecular weight forms of ACTH (37) are reported to decrease in circulation as the ovine fetus nears term (38). In vitro, such isoforms of ACTH have been suggested to antagonize bioactive ACTH but only at a concentration of 1 rig/ml (39). Moreover, fetal hypophysectomy results in basal and ACTH-stimulated plasma concentrations of cortisol that are remarkably similar to those in the intact fetuses (40). Thus, sustained concentrations of cortisol in fetal circulation following removal of all forms of fetal pituitary ACTH does not support a role for high molecular weight isoforms of ACTH in the regulation of cortisol in the ovine fetus at the end of gestation. Factors other than ACTH may account for the most common pattern of constitutive cortisol secretion, i.e. isolated cortisol pulses without coincident ACTH pulses. Prostaglandin Ez (PGEJ stimulates fetal cortisol secretion at a time when the fetal adrenal gland is relatively unresponsive to exogenous ACTH (41), presumably by a direct action on the adrenal (42). Mean plasma concentrations of PGE2 in the fetus and ewe increase as term approaches (43, 44). Indeed, the increase in fetal plasma PGE, concentration may actually precede the prepartum rise in fetal plasma ACTH and cortisol (44). Thus, cortisol pulses could be driven by PGE, or some other placental factor. Neural innervation of the adrenal may also contribute to the regulation of plasma cortisol, since evidence suggests that the splanchnic nerves may modulate cortisol secretion. The adrenal cortex receives an adrenergicafferent projection from the dorsal root ganglia (45, 46). Electrical stimulation of the splanchnic nerves enhances ACTH-induced cortisol secretion in hypophysectomized lambs and calves (47) and dog (48), possibly by increasing 17-hydroxylase activity. An increase in splanchnic nerve activity may explain the observation that adrenal 17hydroxylase activity increases in the fetal sheep after 140 days (48a). Moreover, bilateral transection of the splanchnic nerves before 135 days gestation decreases the rise in plasma cortisol induced by ACTH compared with controls (49). Thus, the splanchnic nerve could contribute to the mechanism that augments cortisol secretion in the ovine fetus near term.
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FETAL
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In the sheep fetus, the role of fetal ACTH in initiating the cortisol surge at parturition may only be permissive, and marked increases in plasma ACTH near term may not be obligatory for the onset of labor. ACTH is required to activate adrenal growth before 116 days of gestation (48a, 51). However, after 116 days gestation, pituitary stalk sectioning of fetal sheep results in spontaneous delivery at or before term (51). Presumably, these stalksectioned fetuses secrete sufficient ACTH to enable fetal adrenal growth since their adrenal glands were enlarged. This is consistent with the findings of Engler et al. (52) where plasma ACTH concentrations increased after hypothalamic disconnection of adult sheep. In addition, fetal ACTH has other functions including the mediation of stress-induced responses such as hemorrhage (52) and acute hypoxemia (54). In summary, the present study demonstrates that in fetal sheep constitutive cortisol and ACTH secretion is pulsatile. Increases in frequency and amplitude of cortisol pulses near delivery appear to underlie the gestational rise in mean plasma concentrations. In contrast, neither the frequency nor amplitude of pulsatile ACTH secretion change near term. Furthermore, a majority of the variance in cortisol concentrations was not explained by immunoreactive ACTH, nor were a majority of cortisol pulses near term coincident with ACTH pulses. Hence, the present results suggest that another factor acting in concert with ACTH may regulate the marked rise in constitutive cortisol secretion which appears to be an integral component of the endocrine mechanism that initiates parturition in the ovine fetus. Acknowledgments We are grateful to Ms. Lela C. Spears for skillful technical assistance and Mrs. Sheila Whitson for typing the manuscript. We thank Dr. John R. G. Challis for cortisol antisera and appreciate the thoughtful discussions with Drs. Paul M. Plotsky and Dennis Engler. Finally, we thank Dr. Grenith Zimmermann for statistical consultation and Dr. Fred J. Karsch for review of the manuscript.
References 1. Bassett JM, Thorburn GD 1969 Foetal plasma corticosteroids and the initiation of narturition in sheen. J Endocrinol 44:285-286 2. Magyar DM, Frihshal D, Elsner CW, Glatz T, Eliot JK, Lowe AH, Buster JE, Nathanielsz PW 1980 Time-trend analysis of plasma cortisol concentrations in the fetal sheep in relation to parturition. Endocrinology 107:155-159 3. Rees L, Jack PMB, Thomas A, Nathanielsz PW 1975 Role of fetal adrenocorticotrophin during parturition in sheep. Nature 253: 274-275 4. Glickman JA, Challis JRG 1980 The changing response pattern of sheep fetal adrenal cells throughout the course of gestation. Endocrinology 106:1371-1376 5. Rose JC, MacDonald AA, Heymann MA, Rudolph AM 1978 Developmental aspects of the pituitary-adrenal axis response to hemorrhagic stress in lamb fetuses in utero. J Clin Invest 61:424-432 6. Rose JC, Meis PJ, Urgan RB, Greiss FC 1982 In uiuo evidence for increased adrenal sensitivity to adrenocorticotrophin-( l-24) in the
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lamb fetus late in gestation. Endocrinology 110:80-85 7. Wood CE. Rudolnh AM 1983 Neeative feedback reaulation of adrenocorticotropm secretion by cortisol in ovine fetises. Endocrinology 112:1930-1936 8. Lye CL, Mitchell BF, Challis JRG 1983 Activation of ovine fetal adrenal function by pulsatile or continuous administration of adrenocorticotropin-(l-24). I. Effects on fetal plasma corticosteroids. Endocrinology 113:770-776 9. Rivier J, Rivier C, Vale W 1984 Synthetic competitive antagonists of corticotropin-releasing factor: effect on ACTH secretion in the rat. Science 224:889-891 10. Negro-Vilar A, Johnston C, Spinedi E, Valenca M, Lopez F 1988 Physiological role of peptides and amines on the regulation of ACTH secretion. Ann NY Acad Sci 512:218-236 11. Caraty A, Grino M, Locatelli A, Oliver C 1988 Secretion of corticotropin releasing factor (CRF) and vasopressin (AVP) into the hypophysial portal blood of conscious, unrestrained rams. Biochem Biophvs Res Commun 155:841-849 12. Engler D, Pham, T, Fullerton MJ, Funder JW, Clarke IJ 1989 Evidence for ultradian secretion of adrenocorticotropin, B-endorphin, and a-melanocyte stimulating hormone by the ovine anterior and intermediate pituitary, Neuroendocrinology 49:349-360 13. Familari M, Smith Al, Smith R, Funder JW 1989 Arginine vasopressin is a much more potent stimulus to ACTH release from ovine anterior pituitary cells than ovine corticotropin-releasing factor. Neuroendocrinology 50:152-157 14. Gambacciami M, Liu JH, Swartz WH, Tueros VS, Rasmussen D, Yen SSC 1987 Intrinsic pulsatility of ACTH release from the human pituitary in uitro. Clin Endocrinol (Oxf) 26:557-563 15. Jones CT 1979 Normal fluctuations in the concentrations of corticosteroid and adrenocorticotrophin in the plasma of foetal and pregnant sheep. Horm Metab Res 11:237-241 16. Challis JRG, Patrick JE, Cross J, Workewych J, Manchester E, Power S 1981 Short-term fluctuations in the concentrations of cortisol and progesterone in fetal plasma, maternal plasma, and amniotic and allantoic fluids from sheep during late pregnancy. Can J Physiol Pharmacol59:261-267 17. Apostolakis EM, Longo LD, Yellon SM 1991 Regulation of basal adrenocorticotrophin and cortisol secretion by arginine vasopressin in the fetal sheep during late gestation. Endocrinology 129: 295-300 18. Durand P, Cathiard AM, Locatelli A, Dazord A, Saez JM 1981 Spontaneous and adrenocorticotropin (ACTH)-induced maturation of the responsiveness of ovine fetal adrenal cells to in vitro stimulation bv ACTH and cholera toxin. Endocrinoloav L_ 109: 2117-2121 19. Manchester EL, Lye SJ, Challis JRG 1983 Activation of ovine fetal adrenal function by pulsatile or continuous administration of adrenocorticotropin-(l-24). I. Effects on adrenal cell responses in uitro. Endocrinology 113:777-782 20. Lincoln DW 1987 Translation of hypothalamic electrical activity into episodic hormone secretion. In: Crowley WF, Hofler JG (eds) The Episodic Secretion of Hormones. John Wiley & Sons, New York, pp 435-458 21. Johl A, Riniker B, Schenkel-Hulliger L 1974 Identity of structure of ovine and bovine ACTH: correction of revised structure of the ovine hormones. FEBS Lett 45:172-174 22. Wilson MG, Nicholson WE, Holscher MA, Sherrell BJ, Mount CD, Orth DN 1982 Proopiolipomelanocortin peptides in normal pituitary, pituitary tumor, and plasma of normal and Cushing’s horses. Endocrinology 110:941-954 R, Karsch FJ 1980 Pulsatile secretion of luteinizing 23 Goodman hormone: differential suppression by ovarian steroids. Endocrinology 107:1286-1290 24 Veldhuis JD, Johnson ML, Seneta E 1991 Analysis of the copulsatility of anterior pituitary hormones. J Clin Endocrinol Metab 73:569-576 25 lranmanesh A, Lizaralde G, Johnson Ml, Veldhuis JD 1989 Circadian, ultradian, and episodic release of B-endorphin in men, and its temporal coupling with cortisol. J Clin Endocrinol Metab 71: 452-463 26. Zar JH 1974 Testing for goodness of fit. In: Biostatistical Analysis.
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OVINE PULSATILE
Prentice-Hall, Englewood Cliffs, pp 54-56 27. Lotering FK, Gilbert RD, Longo LD 1983 Exercise responses in pregnant sheep: blood gases, temperatures, and fetal cardiovascular system. Am J Physiol 55:842-850 28. Norman L, Challis JRG 1987 Dose-dependent effects of arginine vasopressin on endocrine and blood gas response of fetal sheep during the last third of pregnancy. Can J Physiol Pbarmacol 65: 2291-2296 29. Hennessey DP, Coghlan JP, Hardy KJ, Scoggins BA, Wintour EM 1980 The origin of cortisol in the blood of fetal sheep. J Endocrinol 95:71-79 30. Jones CT 1976 Adrenocorticotrophin transformation and degradation in blood. J Endocrinol71:62P-63P 30a.Carnes M, Kalin NH, Lent SJ, Barksdale CM, Brownfield MS 1988 Pulsatile ACTH secretion: variation with time of day and relationship to cortisol. Peptides 9:325-331 31. Gomez RA, Meernik JG, Kuehl WD, Robillard JE 1984 Developmental aspects of the renal response to hemorrhage during fetal life. Pediatr Res 1840-46 32. Alexander DP, Bashore RA, Britton HG, Forsling ML 1974 Maternal and fetal arginine vasopressin in the chronically catheterized sheep. Biol Neonate 25:242-248 33. Pradier P, Davicco MJ, LeFaivre J, Barlet JP, Delost P 1985 Plasma adrenocorticotropbin, cortisol, and aldosterone responses to ovine corticotrophin-releasing factor and vasopressin in sheep. Acta Endocrinol (Copenh) 111:93-100 34. Durand P, Cathiard AM, Morera AM, Dazord A, Saez JM 1981 Maturation of adrenocorticotropin-sensitive adenylate cyclase of ovine fetal adrenal during late pregnancy. Endocrinology 108: 2114-2119 35. Durand P, Catbiard AM, Locatelli A, Saez JM 1982 Modifications of the steroidogenic pathway during spontaneous and adrenocorticotropin-induced maturation of ovine fetal adrenal. Endocrinology 110:500-505 36. Manchester EL, Challis JRG 1982 The effects of adrenocorticotropin, guanylylimidiphosphate, dibutyryl adenosine 3’,5’-monophosphate and exogenous substrates on corticosteroid output by ovine fetal adrenal cells at different times in pregnancy. Endocrinology 111889-895 37. Gasson JC 1975 Steroidogenic activity of high molecular weight forms of corticotronin. Biochemistrv 18:4215-4224 38. Jones CT, RoebuckMM 1980 ACTH peptides and the development of the fetal adrenal. J Steroid Biochem 12:77-82 39. Roebuck MM, Jones CT, Holland D, Silman R 1980 In vitro effects of high molecular weight forms of ACTH on the fetal sheep adrenal. Nature 284:616-618 40. Connors MH, Liggins GC 1980 Adrenocortical responses to adre-
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nocorticotrophin in the hypophysectomized ovine fetus. J Dev Physiol 2:183-190 41. Louis IM, Challis JR, Robinson JS, Thorburn GD 1976 Rapid increase of foetal corticosteroids after prostaglandin E2. Nature 264:797-799 42. Liggins GC, Scroop GC, Haughey KG 1982 Comparison of the effects of prostaglandin E2, prostacyclin and l-24 adrenocorticotrophin on plasma cortisol levels of fetal sheep. J Endocrinol 95: 153-162 43. Fowden AL, Harding R, Ralph MM, Thorburn GD 1987 The nutritional regulation of plasma prostaglandin E concentrations in the fetus and pregnant ewe during late gestation. J Physiol (Land) 394:1-12 44. Thorburn GD, Rice GE 1990 Placental PGE:! and the initiation of parturition in sheep. In: Murray MD (ed) Eicosanoids in Reproduction. CRC Press, Boca Raton, pp 73-86 45. Holzwarth MA, Cunningham LA, Kleitmen N 1987 The role of adrenal nerves in the regulation of adrenocortical functions. Ann NY Acad Sci 512:449-464 46. Kesse WK, Parker TL, Coupland RE 1988 The innervation of the adrenal gland. I. The source of pre- and post-ganglionic nerve fibres to the rat adrenal gland. J Anat 157:33-41 47. Edwards AV, Jones CT, Bloom SR 1986 Reduced adrenal sensitivity to ACTH in lambs with cut splanchnic nerves. J Endocrinol 110:81-89 48. Engeland WC, Gann DW 1989 Splancbnic nerve stimulation modulates steroid stimulation in hypophysectomized dogs. Neuroendocrinology 50:124-129 48a.Challis JRG, Brooks N 1989 Maturation and activation of hypothalamic-pituitary-adrenal function in fetal sheep. Endocr Rev 10: 182-204 49. Myers DA, Robertshaw D, Nathanielsz PW 1990 Effect of bilateral splanchnic nerve section on adrenal function in the ovine fetus. Endocrinology 127:2328-2335 50. Deleted in proof 51. Liggins GC, Fairclough RJ, Grieves SA, Kendall JZ, Knox BS 1973 The mechanism of initiation of parturition in the ewe. Recent Prog Horm Res 29:111-159 52. Eneler D. Pham T. Liu JP. Fullerton MJ. Clarke IJ. Funder JW 1990 Studies of the regulation of the hypbthalamic-pituitary-disconnection. II. Evidence for in uivo ultradian hypersecretion of proopiomelanocortin peptides by the isolated anterior and intermediate pituitary. Endocrinology 127:1956-1966 53. Deleted in proof 54. Akagi K, Challis JRG 1990 Threshold of hormonal and biophysical responses to acute hypoxaemia in fetal sheep at different gestational ages. Can J Physiol Pharmacol 86:549-555
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Endocrinology Copyright 0 1992 by The Endocrine
Vol. 130, No. 5 Printed in U.S.A.
Society
Dissociation of Pulsatile Cortisol and Adrenocorticotropin Secretion in Fetal Late Gestation* EDE AND
MARIE STEVEN
APOSTOLAKIS, M. YELLON
LAWRENCE
D. LONGO,
JOHANNES
Sheep during D. VELDHUIS,
Division of Perinatal Biology, Departments of Physiology, Pediatrics, and Gynecology and Obstetrics, Loma Linda University, School of Medicine, Loma Linda, California 92350; and Division of Endocrinology and Metabolism, Department of Internal Medicine (J.D. V.), University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
ABSTRACT.
In the fetal sheep, plasma cortisol concentrations gradually increase in the last weeks of gestation and abruptly rise during the final 48-72 h preceding birth. To determine if these changes in mean circulating cortisol concentrations result from increased pulsatile secretion and are driven by changes in ACTH pulses, blood samples from five chronically catheterized fetuses were collected every 5 min for 2 h at 133 days gestation and every 4 days thereafter until delivery at 146 + 2 days. Volume was replaced after each blood sample and erythrocytes were returned every 20 min. Plasma cortisol and ACTH secretion were pulsatile in fetuses at all ages. Cortisol pulse frequency increased significantly with gestation from a mean of 2.2 pulses/2 h at 133 days to 4.8 pulses/2 h at 146 days. The interpulse interval (mean + SE) decreased between 133 and 146 days from 54 + 11 min to 23 + 3 min, respectively. Cortisol pulse amplitude increased significantly from 10 + 2 rig/ml at
I
N THE fetal sheep, cortisol in circulation gradually increases over the last third of gestation to abruptly rise during the 72 h preceding birth (1, 2). This surge in fetal cortisol precedes the endocrine cascade which initiates parturition (3, 4). In parallel with changes in cortisol in circulation, fetal plasma ACTH also increases, but less dramatically (5-8). These findings are based on daily and/or less frequent blood sampling protocols. To define the temporal relationship between the secretion of the trophic hormone ACTH and the response of its target organ, the adrenal cortex, more frequent assessment of concentrations in circulation is required. PulsaReceived September 23, 1991. Address all correspondence and requests for reprints to: Steven M. Yellon, Ph.D., Division of Perinatal Biology, Loma Linda University, School of Medicine, Loma Linda, California 92350. * This work was supported by grants from the US Public Health Service (HD-03817 to L. D. L.) and the Center for Nursing Research (NRSA-06042 to E. M. A.), and the Pediatrics Department Research Fund, Loma Linda University School of Medicine. Preliminary work was presented at the 23rd Annual Meeting of the Society for Study of Reproduction, July 17-21, 1990, Knoxville, Tennessee (No. 413).
133 days to 44 + 13 rig/ml at 146 days. In contrast to cortisol, ACTH pulse frequency (3 + 0.6 pulses/2 h) and amplitude (21 + 3 pg/ml) were similar at 133 days and 146 days. The coincidence of cortisol and ACTH pulses did not change between 133 and 146 days. Furthermore, the number of coincident pulses failed to exceed random associations (hypergeometric probability analysis) and could have occurred by chance alone (P values ranged from 0.11-0.63). A point by point comparison of cortisol and ACTH concentrations in fetal circulation indicate that only 36% of the variance in cortisol concentrations could be explained by variance in ACTH (cross-correlation analysis). These data suggest that fetal cortisol and ACTH secretion are pulsatile and that, as gestation advances, increases in constitutive cortisol pulse amplitude and frequency may not be predominantly driven by pulsatile changes in ACTH in the ovine fetal circulation near term. (Endocrinology 130: 2571-2578, 1992)
tile ACTH release occurs in adults of various species, including rats (9), dogs (lo), and sheep (ll-13), as well as from human fetal pituitaries in vitro (14). In the fetal sheep, evidence suggests that concentrations of cortisol and ACTH in the circulation vary within short time intervals (15-17). The goal of the present study was to determine whether plasma cortisol and ACTH secretion is pulsatile in fetal sheep. Since increased adrenal responsiveness to exogenous ACTH stimulation occurs in fetal sheep during late gestation (18, 19) and tissue responsiveness to a trophic hormone can be controlled by patterns of pulsatile secretion (20), we also tested whether increasing fetal cortisol during late gestation was temporally associated with enhanced pulsatile ACTH secretion.
Materials
and Methods
General
Studieswere performed in five time-dated pregnant singleton ewesof mixed Western breed (Nebeker Farms, Santa Monica, 2571
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CA). Ewes experienced 10 h of light each day (lights on 0700 h, PST) in a room with constant humidity and temperature. A dim red light ( 0.05, Kolmogorov-Smirnov test). In summary, less than one third of the variance in cortisol concentrations were correlated to changes in ACTH concentrations, a relationship that is reminiscent of the one third of ACTH pulses that were coincident with peak pulses of cortisol in fetal circulation (Fig. 3), and which was not statistically distinguishable from random occurrence. Discussion
The results provide the first in uiuo evidence that endogenous basal secretion of cortisol and ACTH in fetal sheep is pulsatile during the last 2-3 weeks of gestation. The data indicate that the rise in mean plasma cortisol concentration in the fetus with gestation can be attributed to an increase in both frequency and amplitude of cortisol pulses in circulation, assuming no change in hormone half-life. The results also suggest that this rise in fetal cortisol is not entirely driven by fetal ACTH. Although ACTH secretion is also pulsatile in the ovine fetus, there was little evidence for a change in either pulse frequency or amplitude near term. These data, in TABLE
1. Range
and median
cross-correlation
(r) values
between
plasma
HORMONE
age
-10
cortisol
-5
Range
Median
133
0.01-0.69”
137
0.01-0.56
0.28 0.17
142 146
-0.08-0.76 -0.16-0.23
a P < 0.05 indicates
a nonrandom
0.27 0.012
distribution
Range 0.27-0.80" 0.15-0.73" 0.09-0.78" 0.01-0.21
between
and ACTH
concentration
Range
0.44 0.21 0.48 0.20
plasma
cortisol
in fetal sheep
lag (min) +10
+5
0
Median
2575
conjunction with the finding that a majority of cortisol pulses near delivery are not associated with pulsatile ACTH secretion, suggest that ACTH may not be the principal trophic drive of constitutive pulsatile cortisol secretion in the fetal sheep during the days preceding parturition. An alternative to this interpretation is that a pulse of ACTH provokes a volley of pulses or sustained release of cortisol. Cortisol secretion is enhanced in sheep during the weeks preceding delivery as the fetal adrenal matures (6). In previous studies of fetal sheep older than 135 days gestation, increases in ACTH concentration of 80-100 pg/ml elevated plasma cortisol levels for less than 15 min while ACTH concentrations of more than 500 pg/ml stimulated high cortisol levels for 30 min (17, 28). In the present study of fetuses at 146 days, mean ACTH concentrations and nadir were 60 f 12 and 53 + 6 pg/ml, respectively. Further, on only one occasion during a pulse study did maximal ACTH levels exceed 81 pg/ml. Thus, it seems unlikely in the present study of constitutive secretion that peak episodes in ACTH secretion could account for a pulse volley or sustained release of cortisol. It should be recognized that data derived from blood samples collected at an interval of 5 min over 2 h has the inherent potential to underestimate high frequencylow amplitude pulsatile hormone secretion. We estimate that up to 4 cortisol pulses/2 h and 8 ACTH pulses/2 h could be clearly resolved (maximum amplitude of 15 ng/ ml and 5 pg/ml, respectively) without an upward drift in mean nadir concentration. These estimates are empirically based on the half-life of the respective hormones (29, 30) and the absence of change in nadir hormone levels during the 2-h pulse study. In the present study, cortisol pulse amplitudes were rarely lower than those stated above. In addition, a rise in mean cortisol nadir concentration with gestation was associated with increased cortisol pulse frequency and amplitude. By contrast, the ACTH nadir concentration failed to change with gestational age, and throughout the study, pulse frequency was well below the estimated 8 pulses per 2 h, the upper limit of detection. In addition, ACTH pulse amplitude was comparable to that reported by Engler et al. (12) in adult sheep using a 2-min sampling interval.
Time Gestation (days)
SECRETION
Median
Range
Median
0.01-0.84 0.04-0.70
0.34 0.29
0.25-0.70" 0.08-0.43"
0.54 0.25
0.31-0.75" 0.15-0.35"
0.59 0.36
0.39-0.68" 0.05-0.40
0.49 0.25
and ACTH
concentrations
Range
Median 0.25
0.37-0.70" 0.05-0.62 0.12-0.53"
0.21 0.34 0.08
0.08-0.27
by the Kolmogorov-Smirnov
test (25).
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Moreover, even with a 2-min sampling interval, pulse amplitude may be underestimated. Thus, there is little evidence to suggest that a high frequency-low amplitude pattern of ACTH secretion remained undetected or is biologically relevant for regulating cortisol pulses in the ovine fetus (30a). The potential for fetal stress was another concern in this study. The intensive blood withdrawal protocols required to demonstrate spontaneous variations in constitutive ACTH and cortisol in circulation could be confounded by stress-related secretion. A decrease in total circulating blood volume greater than 7% has been reported to initiate hypothalamic release of arginine vasopressin which, in turn, stimulates release of ACTH and cortisol (31, 32). In the present study, we withdrew less than 3% of circulating volume in total. Moreover, blood volume was immediately replaced and erythrocytes returned every 20 min, in effect virtually no loss in total circulating blood volume. The conclusion that the 5-min blood withdrawal protocol did not stress the fetus or stimulate ACTH release is further supported by the lack of correlation between pulsatile hormone secretion and either the sampling frequency or erythrocyte replacement. In addition, the amplitude of ACTH and cortisol pulses and means of each 2-h pulse study at all gestational ages in the present project were within limits of previous reports (6-8, 15), and typically well below those observed in fetal sheep with mild hypovolemia (31, 32) or after receiving exogenous AVP (15, 33). Finally, arterial blood gases remained stable and within normal range during each pulse study. The crucial question remains what drives the gestational rise in fetal cortisol secretion. There is little to suggest that fetal ACTH is the principal modulator of constitutive cortisol secretion in the present study, because one third or fewer of cortisol pulses were coincident with ACTH pulses during late gestation and a maximum of 36% of the variance in cortisol concentration could be explained by ACTH levels (square of the median r value in Table 1). By contrast, in adult sheep, at least 70% of the recognized cortisol pulses were associated with pulses of ACTH in circulation (12). Others have proposed that the ovine fetal adrenal becomes more sensitive to ACTH as term approaches (6, 34-36). By definition, if adrenal sensitivity to ACTH increased with gestation, an ACTH pulse of similar amplitude and frequency at 133 and 145 days should induce increased cortisol secretion at 146 days. In the present study, amplitude of cortisol pulses increased at 146 days compared with 133 days. Further, coincidence of conjoint ACTH and cortisol pulses were increased while the number of isolated ACTH pulses (those not associated with cortisol pulses) decreased at 146 days compared with 133 days. Thus, the data cannot exclude the possibility that adrenal sensitivity to ACTH
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Endo. Voll30.
1992 No 5
increases with fetal maturation. However, the number of cortisol pulses not associated with ACTH secretion dramatically increased and the distribution of cortisol pulses was more random relative to an ACTH pulse at lag times of +5 min, +lO min (table l), and +15 min (data not shown) near term. Collectively, the data raise the possibility that a trophic factor other than immunoreactive ACTH may drive the marked rise in circulating fetal cortisol at the end of gestation. Removal of an inhibitor of fetal adrenal steroidogenesis may also account for the gestational rise in fetal cortisol. High molecular weight forms of ACTH (37) are reported to decrease in circulation as the ovine fetus nears term (38). In vitro, such isoforms of ACTH have been suggested to antagonize bioactive ACTH but only at a concentration of 1 rig/ml (39). Moreover, fetal hypophysectomy results in basal and ACTH-stimulated plasma concentrations of cortisol that are remarkably similar to those in the intact fetuses (40). Thus, sustained concentrations of cortisol in fetal circulation following removal of all forms of fetal pituitary ACTH does not support a role for high molecular weight isoforms of ACTH in the regulation of cortisol in the ovine fetus at the end of gestation. Factors other than ACTH may account for the most common pattern of constitutive cortisol secretion, i.e. isolated cortisol pulses without coincident ACTH pulses. Prostaglandin Ez (PGEJ stimulates fetal cortisol secretion at a time when the fetal adrenal gland is relatively unresponsive to exogenous ACTH (41), presumably by a direct action on the adrenal (42). Mean plasma concentrations of PGE2 in the fetus and ewe increase as term approaches (43, 44). Indeed, the increase in fetal plasma PGE, concentration may actually precede the prepartum rise in fetal plasma ACTH and cortisol (44). Thus, cortisol pulses could be driven by PGE, or some other placental factor. Neural innervation of the adrenal may also contribute to the regulation of plasma cortisol, since evidence suggests that the splanchnic nerves may modulate cortisol secretion. The adrenal cortex receives an adrenergicafferent projection from the dorsal root ganglia (45, 46). Electrical stimulation of the splanchnic nerves enhances ACTH-induced cortisol secretion in hypophysectomized lambs and calves (47) and dog (48), possibly by increasing 17-hydroxylase activity. An increase in splanchnic nerve activity may explain the observation that adrenal 17hydroxylase activity increases in the fetal sheep after 140 days (48a). Moreover, bilateral transection of the splanchnic nerves before 135 days gestation decreases the rise in plasma cortisol induced by ACTH compared with controls (49). Thus, the splanchnic nerve could contribute to the mechanism that augments cortisol secretion in the ovine fetus near term.
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FETAL
OVINE
PULSATILE
In the sheep fetus, the role of fetal ACTH in initiating the cortisol surge at parturition may only be permissive, and marked increases in plasma ACTH near term may not be obligatory for the onset of labor. ACTH is required to activate adrenal growth before 116 days of gestation (48a, 51). However, after 116 days gestation, pituitary stalk sectioning of fetal sheep results in spontaneous delivery at or before term (51). Presumably, these stalksectioned fetuses secrete sufficient ACTH to enable fetal adrenal growth since their adrenal glands were enlarged. This is consistent with the findings of Engler et al. (52) where plasma ACTH concentrations increased after hypothalamic disconnection of adult sheep. In addition, fetal ACTH has other functions including the mediation of stress-induced responses such as hemorrhage (52) and acute hypoxemia (54). In summary, the present study demonstrates that in fetal sheep constitutive cortisol and ACTH secretion is pulsatile. Increases in frequency and amplitude of cortisol pulses near delivery appear to underlie the gestational rise in mean plasma concentrations. In contrast, neither the frequency nor amplitude of pulsatile ACTH secretion change near term. Furthermore, a majority of the variance in cortisol concentrations was not explained by immunoreactive ACTH, nor were a majority of cortisol pulses near term coincident with ACTH pulses. Hence, the present results suggest that another factor acting in concert with ACTH may regulate the marked rise in constitutive cortisol secretion which appears to be an integral component of the endocrine mechanism that initiates parturition in the ovine fetus. Acknowledgments We are grateful to Ms. Lela C. Spears for skillful technical assistance and Mrs. Sheila Whitson for typing the manuscript. We thank Dr. John R. G. Challis for cortisol antisera and appreciate the thoughtful discussions with Drs. Paul M. Plotsky and Dennis Engler. Finally, we thank Dr. Grenith Zimmermann for statistical consultation and Dr. Fred J. Karsch for review of the manuscript.
References 1. Bassett JM, Thorburn GD 1969 Foetal plasma corticosteroids and the initiation of narturition in sheen. J Endocrinol 44:285-286 2. Magyar DM, Frihshal D, Elsner CW, Glatz T, Eliot JK, Lowe AH, Buster JE, Nathanielsz PW 1980 Time-trend analysis of plasma cortisol concentrations in the fetal sheep in relation to parturition. Endocrinology 107:155-159 3. Rees L, Jack PMB, Thomas A, Nathanielsz PW 1975 Role of fetal adrenocorticotrophin during parturition in sheep. Nature 253: 274-275 4. Glickman JA, Challis JRG 1980 The changing response pattern of sheep fetal adrenal cells throughout the course of gestation. Endocrinology 106:1371-1376 5. Rose JC, MacDonald AA, Heymann MA, Rudolph AM 1978 Developmental aspects of the pituitary-adrenal axis response to hemorrhagic stress in lamb fetuses in utero. J Clin Invest 61:424-432 6. Rose JC, Meis PJ, Urgan RB, Greiss FC 1982 In uiuo evidence for increased adrenal sensitivity to adrenocorticotrophin-( l-24) in the
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lamb fetus late in gestation. Endocrinology 110:80-85 7. Wood CE. Rudolnh AM 1983 Neeative feedback reaulation of adrenocorticotropm secretion by cortisol in ovine fetises. Endocrinology 112:1930-1936 8. Lye CL, Mitchell BF, Challis JRG 1983 Activation of ovine fetal adrenal function by pulsatile or continuous administration of adrenocorticotropin-(l-24). I. Effects on fetal plasma corticosteroids. Endocrinology 113:770-776 9. Rivier J, Rivier C, Vale W 1984 Synthetic competitive antagonists of corticotropin-releasing factor: effect on ACTH secretion in the rat. Science 224:889-891 10. Negro-Vilar A, Johnston C, Spinedi E, Valenca M, Lopez F 1988 Physiological role of peptides and amines on the regulation of ACTH secretion. Ann NY Acad Sci 512:218-236 11. Caraty A, Grino M, Locatelli A, Oliver C 1988 Secretion of corticotropin releasing factor (CRF) and vasopressin (AVP) into the hypophysial portal blood of conscious, unrestrained rams. Biochem Biophvs Res Commun 155:841-849 12. Engler D, Pham, T, Fullerton MJ, Funder JW, Clarke IJ 1989 Evidence for ultradian secretion of adrenocorticotropin, B-endorphin, and a-melanocyte stimulating hormone by the ovine anterior and intermediate pituitary, Neuroendocrinology 49:349-360 13. Familari M, Smith Al, Smith R, Funder JW 1989 Arginine vasopressin is a much more potent stimulus to ACTH release from ovine anterior pituitary cells than ovine corticotropin-releasing factor. Neuroendocrinology 50:152-157 14. Gambacciami M, Liu JH, Swartz WH, Tueros VS, Rasmussen D, Yen SSC 1987 Intrinsic pulsatility of ACTH release from the human pituitary in uitro. Clin Endocrinol (Oxf) 26:557-563 15. Jones CT 1979 Normal fluctuations in the concentrations of corticosteroid and adrenocorticotrophin in the plasma of foetal and pregnant sheep. Horm Metab Res 11:237-241 16. Challis JRG, Patrick JE, Cross J, Workewych J, Manchester E, Power S 1981 Short-term fluctuations in the concentrations of cortisol and progesterone in fetal plasma, maternal plasma, and amniotic and allantoic fluids from sheep during late pregnancy. Can J Physiol Pharmacol59:261-267 17. Apostolakis EM, Longo LD, Yellon SM 1991 Regulation of basal adrenocorticotrophin and cortisol secretion by arginine vasopressin in the fetal sheep during late gestation. Endocrinology 129: 295-300 18. Durand P, Cathiard AM, Locatelli A, Dazord A, Saez JM 1981 Spontaneous and adrenocorticotropin (ACTH)-induced maturation of the responsiveness of ovine fetal adrenal cells to in vitro stimulation bv ACTH and cholera toxin. Endocrinoloav L_ 109: 2117-2121 19. Manchester EL, Lye SJ, Challis JRG 1983 Activation of ovine fetal adrenal function by pulsatile or continuous administration of adrenocorticotropin-(l-24). I. Effects on adrenal cell responses in uitro. Endocrinology 113:777-782 20. Lincoln DW 1987 Translation of hypothalamic electrical activity into episodic hormone secretion. In: Crowley WF, Hofler JG (eds) The Episodic Secretion of Hormones. John Wiley & Sons, New York, pp 435-458 21. Johl A, Riniker B, Schenkel-Hulliger L 1974 Identity of structure of ovine and bovine ACTH: correction of revised structure of the ovine hormones. FEBS Lett 45:172-174 22. Wilson MG, Nicholson WE, Holscher MA, Sherrell BJ, Mount CD, Orth DN 1982 Proopiolipomelanocortin peptides in normal pituitary, pituitary tumor, and plasma of normal and Cushing’s horses. Endocrinology 110:941-954 R, Karsch FJ 1980 Pulsatile secretion of luteinizing 23 Goodman hormone: differential suppression by ovarian steroids. Endocrinology 107:1286-1290 24 Veldhuis JD, Johnson ML, Seneta E 1991 Analysis of the copulsatility of anterior pituitary hormones. J Clin Endocrinol Metab 73:569-576 25 lranmanesh A, Lizaralde G, Johnson Ml, Veldhuis JD 1989 Circadian, ultradian, and episodic release of B-endorphin in men, and its temporal coupling with cortisol. J Clin Endocrinol Metab 71: 452-463 26. Zar JH 1974 Testing for goodness of fit. In: Biostatistical Analysis.
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Prentice-Hall, Englewood Cliffs, pp 54-56 27. Lotering FK, Gilbert RD, Longo LD 1983 Exercise responses in pregnant sheep: blood gases, temperatures, and fetal cardiovascular system. Am J Physiol 55:842-850 28. Norman L, Challis JRG 1987 Dose-dependent effects of arginine vasopressin on endocrine and blood gas response of fetal sheep during the last third of pregnancy. Can J Physiol Pbarmacol 65: 2291-2296 29. Hennessey DP, Coghlan JP, Hardy KJ, Scoggins BA, Wintour EM 1980 The origin of cortisol in the blood of fetal sheep. J Endocrinol 95:71-79 30. Jones CT 1976 Adrenocorticotrophin transformation and degradation in blood. J Endocrinol71:62P-63P 30a.Carnes M, Kalin NH, Lent SJ, Barksdale CM, Brownfield MS 1988 Pulsatile ACTH secretion: variation with time of day and relationship to cortisol. Peptides 9:325-331 31. Gomez RA, Meernik JG, Kuehl WD, Robillard JE 1984 Developmental aspects of the renal response to hemorrhage during fetal life. Pediatr Res 1840-46 32. Alexander DP, Bashore RA, Britton HG, Forsling ML 1974 Maternal and fetal arginine vasopressin in the chronically catheterized sheep. Biol Neonate 25:242-248 33. Pradier P, Davicco MJ, LeFaivre J, Barlet JP, Delost P 1985 Plasma adrenocorticotropbin, cortisol, and aldosterone responses to ovine corticotrophin-releasing factor and vasopressin in sheep. Acta Endocrinol (Copenh) 111:93-100 34. Durand P, Cathiard AM, Morera AM, Dazord A, Saez JM 1981 Maturation of adrenocorticotropin-sensitive adenylate cyclase of ovine fetal adrenal during late pregnancy. Endocrinology 108: 2114-2119 35. Durand P, Catbiard AM, Locatelli A, Saez JM 1982 Modifications of the steroidogenic pathway during spontaneous and adrenocorticotropin-induced maturation of ovine fetal adrenal. Endocrinology 110:500-505 36. Manchester EL, Challis JRG 1982 The effects of adrenocorticotropin, guanylylimidiphosphate, dibutyryl adenosine 3’,5’-monophosphate and exogenous substrates on corticosteroid output by ovine fetal adrenal cells at different times in pregnancy. Endocrinology 111889-895 37. Gasson JC 1975 Steroidogenic activity of high molecular weight forms of corticotronin. Biochemistrv 18:4215-4224 38. Jones CT, RoebuckMM 1980 ACTH peptides and the development of the fetal adrenal. J Steroid Biochem 12:77-82 39. Roebuck MM, Jones CT, Holland D, Silman R 1980 In vitro effects of high molecular weight forms of ACTH on the fetal sheep adrenal. Nature 284:616-618 40. Connors MH, Liggins GC 1980 Adrenocortical responses to adre-
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nocorticotrophin in the hypophysectomized ovine fetus. J Dev Physiol 2:183-190 41. Louis IM, Challis JR, Robinson JS, Thorburn GD 1976 Rapid increase of foetal corticosteroids after prostaglandin E2. Nature 264:797-799 42. Liggins GC, Scroop GC, Haughey KG 1982 Comparison of the effects of prostaglandin E2, prostacyclin and l-24 adrenocorticotrophin on plasma cortisol levels of fetal sheep. J Endocrinol 95: 153-162 43. Fowden AL, Harding R, Ralph MM, Thorburn GD 1987 The nutritional regulation of plasma prostaglandin E concentrations in the fetus and pregnant ewe during late gestation. J Physiol (Land) 394:1-12 44. Thorburn GD, Rice GE 1990 Placental PGE:! and the initiation of parturition in sheep. In: Murray MD (ed) Eicosanoids in Reproduction. CRC Press, Boca Raton, pp 73-86 45. Holzwarth MA, Cunningham LA, Kleitmen N 1987 The role of adrenal nerves in the regulation of adrenocortical functions. Ann NY Acad Sci 512:449-464 46. Kesse WK, Parker TL, Coupland RE 1988 The innervation of the adrenal gland. I. The source of pre- and post-ganglionic nerve fibres to the rat adrenal gland. J Anat 157:33-41 47. Edwards AV, Jones CT, Bloom SR 1986 Reduced adrenal sensitivity to ACTH in lambs with cut splanchnic nerves. J Endocrinol 110:81-89 48. Engeland WC, Gann DW 1989 Splancbnic nerve stimulation modulates steroid stimulation in hypophysectomized dogs. Neuroendocrinology 50:124-129 48a.Challis JRG, Brooks N 1989 Maturation and activation of hypothalamic-pituitary-adrenal function in fetal sheep. Endocr Rev 10: 182-204 49. Myers DA, Robertshaw D, Nathanielsz PW 1990 Effect of bilateral splanchnic nerve section on adrenal function in the ovine fetus. Endocrinology 127:2328-2335 50. Deleted in proof 51. Liggins GC, Fairclough RJ, Grieves SA, Kendall JZ, Knox BS 1973 The mechanism of initiation of parturition in the ewe. Recent Prog Horm Res 29:111-159 52. Eneler D. Pham T. Liu JP. Fullerton MJ. Clarke IJ. Funder JW 1990 Studies of the regulation of the hypbthalamic-pituitary-disconnection. II. Evidence for in uivo ultradian hypersecretion of proopiomelanocortin peptides by the isolated anterior and intermediate pituitary. Endocrinology 127:1956-1966 53. Deleted in proof 54. Akagi K, Challis JRG 1990 Threshold of hormonal and biophysical responses to acute hypoxaemia in fetal sheep at different gestational ages. Can J Physiol Pharmacol 86:549-555
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