0013-7227/91/1294-1942$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 129, No. 4 Printed in U.S.A.

Effect of Adrenalectomy or Long Term Cortisol or Adrenocorticotropin (ACTH)-Releasing Factor Infusion on the Concentration and Molecular Weight Distribution of ACTH in Fetal Sheep Plasma* I. Z. OZOLINS, G. C. ANTOLOVICH, C. A. BROWNE, R. A. PERRY, P. M. ROBINSON, M. SILVER, AND I. C. McMILLEN Department of Physiology (I.Z.O., C.A.B., I.C.M.), Monash University, Clayton, Victoria 3168; Department of Anatomy (G.C.A., R.A.P., P.M.R.), University of Melbourne, Parkville, Victoria, Australia 3052; and Physiological Laboratory (M.S.), University of Cambridge, Cambridge United Kingdom

139 days (26.8 ± 9.3%) but not different to that after 140 days gestation (29.9 ± 5.5%). Between 110-117 days, cortisol infusion had no effect on the proportion of IR-ACTH in the LMW range (43.9 ± 5.7%, saline infused; 44.1 ± 2.4%, cortisol infused). Between 121-125 days, the proportion of IR-ACTH in the LMW range in the CRF-infused groups (with or without prior exposure to cortisol) was significantly lower (27.4 ± 2.1%) than in the saline-infused control group. In contrast, after fetal adrenalectomy, the proportion of IR-ACTH in the LMW range between 126-139 days was significantly higher (48.0 ± 6.7%) than in intact control animals (23.8 ± 3.5%). We conclude that the change in the mol wt profile of IRACTH in fetal plasma after 125 days may be a consequence of changes in the morphological and/or functional characteristics of the corticotrophic cells in the fetal pituitary. Infusion of oCRF appears to accelerate the normal maturation of the fetal pituitary-adrenal relationship, and oCRF acting either directly or via secretion of cortisol may play a role in the posttranslational processing of POMC in the fetal sheep pituitary after 125 days gestation. {Endocrinology 129:1942-1950,1991)

ABSTRACT. It is unclear whether the maturation of corticotrophs from the fetal to the adult type in the fetal sheep pituitary in late gestation is associated with changes in the sensitivity of the fetal pituitary to corticotrophic secretagogues and in the form of ACTH-containing peptides (IR-ACTH) secreted into the circulation. The maturation of the pituitary corticotroph population is known to be accelerated by intrafetal cortisol infusion and delayed by bilateral fetal adrenalectomy. We have therefore investigated the mol wt profile of IR-ACTH present in fetal sheep plasma from 110 days gestation until term (147 ± 3 days) and determined whether intrafetal cortisol infusion between 105-117 days (2.5 mg cortisol/day), or bilateral fetal adrenalectomy can alter the mol wt profile of IR-ACTH in fetal sheep plasma. We have also investigated whether prior exposure to cortisol alters the subsequent responsiveness of the fetal pituitary to a long term infusion of ovine (o) CRF (10 pg oCRF/day). In the control group, the proportion of IR-ACTH which eluted in the low-mol wt (LMW) range (i.e. 140 Days oCRF-infused 118-125 Days 126-139 Days

HMW IRACTH (ng/1)

( o)

LMW IRACTH (ng/1)

91.4 ± 9.6° 85.4 ± 12.7° 357.3 ± 101.6*

43.9 ± 4.2* 26.8 ± 9.3° 29.9 ± 5.5°-*

40.7 ± 7.4O>* 19.6 ± 3.5" 122.8 ± 56.8*

56.2 ± 4.2° 73.2 ± 9.3* 70.1 ± 5.5"-*

50.7 ±5.1° 65.8 ± 15.0° 234.6 ± 45.6*

253.5 ± 91.3* 294.3 ± 86.5*

27.4 ± 2.1° 32.7 ± 2.9°-*

75.4 ± 33.5* 102.3 ± 29.1*

72.6 ± 1.9* 67.3 ± 2.9°'*

178.2 ± 58.0* 192.0 ± 42.6*

Total IRACTH (ng/1)

LMW

HMW o)

The mean (±SEM) total IR-ACTH concentration (ng/1), proportion of total IR-ACTH in the low mol wt (LMW) form (%), total LMW IRACTH concentration (ng/1), proportion of total IR-ACTH in the high mol wt (HMW) form (%), and total HMW IR-ACTH concentration (ng/1) in plasma from fetal sheep in the control (SAL + SAL) group at 118-125 days (n = 4), 126-139 days (n = 4), and 140-147 days (n = 4) gestation and in the oCRF-infused (SAL + CRF and F + CRF) groups at 118-125 days (n = 4) and 126-139 days gestation (n = 6). ab

' Different superscripts in each column denote values that are significantly different (p < 0.05) from each other.

i2oT a.

VACTH

INTACT

80-

40 •

40

80

120

160

200

ELUTION VOLUME (ml)

FIG. 4. The chromatographic profile of IR-ACTH in the plasma of a, an intact fetus and b, an ADRX fetus at 130 days. The proportion of IR-ACTH which elutes in the LMW range is greater in the ADRX fetus. The arrows mark the Vo and Vt of the column and the Ve of [125I] ACTH.

correlated from as early as 118-125 days gestation. This acceleration of the normal maturation of the fetal pituitary-adrenal relationship would appear to be a consequence of the significant increase in the plasma concen-

trations of IR-ACTH, which occurred immediately after the onset of the oCRF infusion. Plasma IR-ACTH and cortisol concentrations remained elevated throughout the whole oCRF infusion period, suggesting that there was no down-regulation during the long term continuous administration of oCRF. A recent report (19) described the effects of pulsatile intrafetal administration of oCRF (1 /ig/4 h for 7 days) on the fetal pituitary and adrenal responses from around 120 days gestation. As in the present study, the oCRF treatment significantly increased basal IR-ACTH and cortisol concentrations on all days of treatment. Therefore, either continuous or pulsatile administration of oCRF at 110-120 days gestation can activate the fetal pituitary-adrenal axis to increase plasma IR-ACTH and cortisol concentrations to levels normally only found immediately prepartum. Although it was not the aim of the present study to induce premature parturition, four of the ewes in the CRF-infused groups did deliver prematurely at 130-135 days. We observed a greater incidence in fetal deaths in the CRF-infused groups (three out of nine), compared to the saline-infused group (zero out of five). These deaths occurred in the absence of any deleterious change in fetal blood gas status, but the fetal hemoglobin and hematocrit were both significantly reduced during the CRF infusion period. These changes may be a consequence of the increased cortisol concentrations in the CRF-infused group, but whether they lead to the increased fetal vulnerability is unclear. Antolovich and coworkers (10) have demonstrated that infusion of cortisol (at 2 mg/24 h) for 6 days from 109 days gestation caused a significant increase in the proportion of adult corticotrophs and a corresponding decrease in the proportion of fetal corticotrophs present in the fetal sheep pituitary at 115 days gestation. The mean percentages of adult and fetal corticotrophs in the cortisol-infused fetal sheep at 115 days gestation were sim-

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ADRENALECTOMY, CORTISOL, AND CRF ON FETAL ACTH

ilar to those in normal fetal sheep pituitaries at 135 days gestation. Although the results of the present study suggest that an increase in the proportion of adult corticotrophs as a result of prior cortisol exposure is not associated with a change in the pituitary responsiveness to oCRF, it is also possible that the effect of cortisol on the maturation of fetal corticotrophs is more rapid than established in the previous study. Plasma cortisol concentrations increased within 24 h of the start of the oCRF infusion in the SAL + CRF group, and it may be that changes in the morphological appearance of the corticotrophic cell types may occur after only 24-48 h exposure to high cortisol concentrations. Collection of fetal pituitaries for morphological examination within 48 h of the start of an oCRF or cortisol infusion at 117 days gestation would be required to establish whether this was the case. The normal morphological maturation of the corticotroph in the fetal pituitary may be associated with a change in the posttranslational processing of the ACTH precursor POMC (10). We have demonstrated that ACTH-containing peptides were present in mainly two mol wt ranges, i.e. less than or more than 12 K, and that there were changes in the relative proportions of these different mol wt forms of ACTH throughout the period in late gestation when the change in the predominance of fetal to adult corticotroph occurs in the fetal pituitary. Jones and Roebuck (6) reported that there was an increase in the ratio of the amount of 4.5 K ACTH (i.e. bioactive ACTH 1-39) to higher mol wt forms of ACTH in fetal sheep plasma after 138 days gestation. In our study, the major change in the distribution of ACTHcontaining peptides in fetal plasma occurred at around 125 days gestation, when there was a significant fall in the proportion of IR-ACTH present in the LMW range. Changes in the pattern of posttranslational processing of the ACTH precursor, POMC, could be a result of a change in the corticotrophic cell type in the fetal pituitary. It is interesting that the fetal cell is the predominant corticotroph present in the pituitary of the normal fetus before 125 days and in the adrenalectomized fetus at 135 days, i.e. when there is the highest proportion of IRACTH in the LMW range in the fetal circulation. Although we have described morphological heterogeneity of corticotrophic cells in the fetal pituitary, there are also reports of functional heterogeneity of corticotrophic cells in the adult pituitary (20, 21). Populations of corticotrophs which are predominantly vasopressin or CRF responsive have been described (20). In this context it is noteworthy that before 125 days the fetal sheep pituitary is more responsive to vasopressin than CRF (22) and that in the adrenalectomized rat the AVP:CRF molar ratio increases in the hypophyseal portal circulation (23). It may be that the vasopressin responsive corticotrophs

1949

secrete predominantly more LMW ACTH either in the basal (i.e. before 125 days gestation) or stimulated (i.e. adrenalectomized) state. Whether the fetal-type corticotrophs are predominantly vasopressin responsive clearly needs to be established. Infusion of oCRF from 117 days caused a significant decrease in the proportion of IR-ACTH in the LMW range and appeared to advance the normal gestational changes in the mol wt profile of IR-ACTH-containing peptides. One possibility is that the decrease in the proportion of IR-ACTH in the LMW form after 125 days gestation in the control fetus is associated with a change from predominantly vasopressin- to CRF-responsive corticotrophs in the fetal pituitary. Intrafetal infusion of oCRF before 125 days gestation could act via recruitment and stimulation of such CRF-responsive corticotrophs. An alternative possibility is that it may be the adrenal (via the secretion of cortisol) rather than the hypothalamus which plays the major role in the regulation of posttranslational processing of POMC in the intact sheep fetus after 125 days gestation and in the oCRFinfused groups. The failure of cortisol alone to alter the mol wt profile of ACTH-containing peptides in fetal sheep plasma before 118 days suggests however that such an action of cortisol on the processing of POMC is dependent on the exposure of the fetal corticotroph to a permissive signal which occurs in the normal fetus after 125 days gestation. Although this study has not identified endogenous CRF as the mediator of the normal prepartum changes in the fetal pituitary-adrenal axis, it has provided evidence that oCRF is capable of generating such changes in the concentrations of ACTH and in the mol wt distribution of ACTH-containing peptides in fetal plasma during late gestation. Further, we have proposed that the changes in the plasma concentrations of IR-ACTH and in the mol wt distribution of IR-ACTH in the late-gestation sheep fetus are a consequence of changes in the functional characteristics of the corticotrophic cells which are associated with changes in the morphological appearance of the corticotrophic cell population.

Acknowledgments We are grateful to Toni Harris, Susan Walmsley, and Campbell York for their expert technical assistance and to David Caddy for his advice on statistical analysis of the data.

References 1. Perry RA, Mulvogue HM, McMillen IC, Robinson PM 1985 Immunohistochemical localization of ACTH in the adult and fetal sheep pituitary. J Dev Physiol 7:397-404 2. Mulvogue HM, McMillen IC, Robinson PM, Perry RA 1986 Immunocytochemical localization of pro-7MSH, 7-MSH, ACTH and /3-endorphin/jS-lipotrophin in fetal sheep pituitary: an ontogenetic

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ADRENALECTOMY, CORTISOL, AND CRF ON FETAL ACTH

study. J Dev Physiol 8:355-368 3. Robinson PM, Rowe EJ, Wintour EM 1979 The histogenesis of the adrenal cortex in the foetal sheep. Acta Endocrinol (Copenh) 91:134-149 4. Bassett JM, Thorburn GD 1969 Foetal plasma corticosteroids and the initiation of parturition in sheep. J Endocrinol 44:285-286 5. Norman LJ, Lye SJ, Wlodek ME, Challis JRG 1985 Changes in pituitary responses to synthetic ovine corticotrophin releasing factor in fetal sheep. Can J Physiol Pharmacol 63:1398-1403 6. Jones CT, Roebuck MM 1980 ACTH peptides and the development of the fetal adrenal. J Steroid Biochem 12:77-82 7. Silman RE, Holland D, Chard T, Lowry PJ, Hope J, Rees LH, Thomas A, Nathanielsz PW 1979 Adrenocorticotrophin-related peptides in adult and foetal sheep pituitary glands. J Endocrinol 81:19-34 8. Brieu V, Durand P 1989 Adrenocorticotropic hormone released by pituitary cells from ovine fetuses and lambs. Polymorphism and biological activity. Neuroendocrinology 49:300-308 9. Pradier P, Dalle M, Tournaire C, Delost P 1988 Plasma concentrations of adrenocorticotropin-related peptides after corticotropinreleasing hormone and vasopressin injections in sheep. Acta Endocrinol (Copenh) 119:391-396 10. Antolovich GC, Perry RA, Trahair JF, Silver M, Robinson PM 1989 The development of corticotrophs in the fetal sheep pars distalis: the effect of adrenalectomy or cortisol infusion. Endocrinology 124:1333-1339 11. Barnes RJ, Comline RS, Silver M 1977 The effects of bilateral adrenalectomy or hypophysectomy of the foetal lamb in utero. J Physiol 264:427-447 12. McMillen IC, Antolovich GC, Mercer JE, Perry RA, Silver M 1990 Proopiomelanocortin messenger RNA levels are increased in the anterior pituitary of the sheep fetus after adrenalectomy in late gestation. Neuroendocrinology 52:297-302 13. Bocking AD, McMillen IC, Harding R, Thorburn GD 1986 Effect of reduced uterine blood flow on fetal and maternal cortisol. J Dev

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Physiol 8:237-245 14. Zemdegs IZ, McMillen IC, Walker DW, Thorburn GD, Nowak R 1988 Diurnal rhythms in plasma melatonin concentrations in the fetal sheep and pregnant ewe during late gestation. Endocrinology 123:284-289 15. Magyar DM, Fridshal D, Eisner CW, Glatz T, Eliot J, Klein AH, Lowe KC, Buster JE, Nathanielsz PW 1980 Time-trend analysis of plasma cortisol concentrations in the fetal sheep in relation to parturition. Endocrinology 107:155-159 16. Hennessy DP, Coghlan JP, Hardy KJ, Wintour EM 1982 Development of the pituitary-adrenal axis in chronically cannulated ovine fetuses. J Dev Physiol 4:339-352 17. Jones CT 1979 Normal fluctuations in the concentration of corticosteroid and adrenocorticotrophin in the plasma of foetal and pregnant sheep. Horm Metab Res 11:237-241 18. Engler D, Pham T, Fullerton MJ, Ooi G, Funder JW, Clarke IJ 1989 Studies of the secretion of corticotropin-releasing factor and arginine vasopressin into the hypophysial-portal circulation of the conscious sheep. I. Effect of an audiovisual stimulus and insulininduced hypoglycemia. Neuroendocrinology 49:367-381 19. Brooks AN, Challis JRG, Norman LJ 1987 Pituitary and adrenal responses to pulsatile ovine corticotropin-releasing factor administered to fetal sheep. Endocrinology 120:2383-2388 20. Childs G-V, Unabin G, Burke JA, Marchetti C 1987 Secretion from corticotropes after avidin-fluorescein stains for biotinylated ligands (CRF or AVP). Am J Physiol 252:E347-E352 21. Schwartz J 1990 Evidence for intrapituitary intercellular control of adrenocorticotropin secretion. Mol Cell Endocrinol 68:77-83 22. Norman LJ, Challis JRG 1987 Synergism between corticotropinreleasing factor and arginine vasopressin on adrenocorticotropin release in vivo varies as a function of gestational age in the ovine fetus. Endocrinology 120:1052-1058 23. Holmes MC, Antoni FA, Catt KJ, Aguilera C 1986 Predominant release of vasopressin vs. corticotropin releasing factor from the isolated median eminence after adrenalectomy. Neuroendocrinology 43:425-430

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Effect of adrenalectomy or long term cortisol or adrenocorticotropin (ACTH)-releasing factor infusion on the concentration and molecular weight distribution of ACTH in fetal sheep plasma.

It is unclear whether the maturation of corticotrophs from the fetal to the adult type in the fetal sheep pituitary in late gestation is associated wi...
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