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MS 8658 Received 24 August 1998; accepted after revision 25 January 1999.
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ABSTRACT |
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INTRODUCTION |
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In the sheep, an intact hypothalamo-pituitary-adrenal axis and a prepartum increase in the fetal plasma concentration of cortisol are required for parturition to occur at 147 ± 3 days gestation (Challis & Brooks, 1989). It has been demonstrated that there is morphological heterogeneity of corticotrophs in the anterior lobe of the fetal sheep pituitary in late gestation (Perry et al. 1985; Mulvogue et al. 1986). The main corticotrophic cell observed between 90 and 130 days gestation is a tall, columnar 'fetal' cell type whereas after 135 days gestation a small, stellate or 'adult' cell predominates. Infusion of cortisol (2 mg day-1) into the fetus between 109 and 115 days gestation results in a premature maturation of the corticotroph population in the fetal pituitary to a predominantly 'adult' type corticotroph (Antolovich et al. 1989). It is unknown whether the morphological heterogeneity in the corticotrophic cell population in the fetal pituitary is associated with a functional heterogeneity which may be important in the prepartum stimulation of the fetal adrenal cortex. Studies using reverse haemolytic plaque assays or a specific cytotoxic analogue of corticotrophin releasing hormone (CRH) have shown that there are separate subpopulations of corticotrophic cells, distinguishable on the basis of response to hypothalamic secretagogues (Neill et al. 1987; Schwartz & Vale, 1988; Jia et al. 1991) in the adult pituitary. In the adult rat, cow and sheep pituitary there are corticotrophs which are responsive to either CRH or arginine vasopressin (AVP) alone or to both CRH and AVP (Neill et al. 1987; Schwartz & Vale, 1988; Jia et al. 1991). Interestingly, there appear to be major functional differences between these cell types in the synthetic and secretory pathways for adrenocorticotrophic hormone (ACTH) and in their responsiveness to glucocorticoids (Schwartz et al. 1994).
In the present study, we have investigated whether there are different subpopulations of corticotrophs in the fetal sheep pituitary and whether there is any change in the functional corticotrophic cell types during late gestation. We have also investigated the effect of intrafetal cortisol infusion on the ACTH synthetic and secretory capacity of the specific corticotrophic cell types. We have cultured pituitary cells collected from fetal sheep in early gestation (116 days), after a 7 day infusion of either saline or cortisol, and in late gestation (140-145 days gestation). During culture we pretreated half the cells from each pituitary with a cytotoxic CRH conjugate, Cx. Cx consists of an analogue of CRH coupled to a plant toxin, gelonin, and acts selectively to kill all CRH target cells (i.e. corticotrophs that respond only to CRH or those that respond to both CRH and AVP). Corticotrophs which only respond to AVP are resistant to the effects of Cx and are spared (Schwartz et al. 1987). We have determined the proportion of ACTH stored and secreted by CRH-responsive cells (i.e. Cx-sensitive cells) in late gestation and after cortisol infusion. We have also determined the effects of gestational age and cortisol infusion on the ACTH responsiveness of corticotrophs to the hypothalamic secretagogues CRH and AVP in the presence and absence of the CRH-cytotoxin.
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METHODS |
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Animals and surgery
All experiments in the study were carried out according to the guidelines of the Standing Committee of Ethics and Animal Experimentation at the University of Adelaide. Twenty-seven dated pregnant Merino × Border Leicester ewes were used in these studies. Surgery was performed in 21 ewes at around 103 days gestation under aseptic conditions with general anaesthesia induced by intravenous sodium thiopentone (Pentothal, 50 mg ml-1; Rhone Merieux Australia Pty Ltd, Qld, Australia) and maintained with 3-4 % halothane in oxygen as previously described (Ozolins et al. 1991). Catheters were inserted into a carotid artery and jugular vein of the fetus and ewe, and into the amniotic cavity, filled with sterile heparinized saline, and exteriorized via an incision in the ewe's flank. All ewes and fetal sheep received 2 ml I.M. ilium penstrep (procaine penicillin, 250 mg ml-1; dihydrostreptomycin sulphate, 250 mg ml-1; procain hydrochloride, 20 mg ml-1: Troy Laboratories, Smithfield, NSW, Australia) at the time of surgery. After a postoperative recovery period, cortisol (2-3 mg 24 h-1 I.V., hydrocortisone succinate, Solucortef; Upjohn, Kalamazoo, MI, USA; n = 11) or saline (4·4 ml 24 h-1 I.V.; n = 10) was infused into the fetus between 109 and 116 days gestation. Fetal (5 ml) and maternal (5 ml) arterial blood samples were collected twice daily (10.00 and 17.00 h) throughout the infusion period into chilled tubes containing lithium heparin (125 i.u.; Disposable Products, Adelaide, South Australia, Australia) and aprotinin (1000 KIU (ml blood)-1; Sigma). All blood samples were centrifuged and plasma was separated into aliquots and stored at -20°C for cortisol radioimmunoassay. Arterial blood samples (0·5 ml) were also collected for measurement of fetal blood status using an ABL 520 acid/base analyser (Radiometer, Copenhagen, Denmark). At 116 days gestation, ewes were killed with an intravenous overdose of sodium pentobarbitone (25 ml, 325 mg ml-1, Lethobarb; Syntex, Castle Hill, NSW, Australia). The fetal sheep were anaesthetized by the maternal overdose of sodium pentobarbitone and delivered via laparotomy, weighed and then killed by decapitation. A separate group of six pregnant ewes and their 10 fetuses were also killed by the same method between 140 and 145 days gestation. Pituitary glands were quickly removed from the fetuses and immediately placed into Hepes dissociation buffer (HDB (mM): NaCl, 137; HCl, 5; Na2HPO4, 0·7; Hepes, 25; pH 7·36).
Pituitary preparation
The anterior (AP) and the neurointermediate lobes (NIL) of each pituitary were gently separated by blunt dissection. The AP tissue was minced into small tissue fragments (
1 mm3) using two scalpel blades. The fragments were washed in HDB solution, and then placed in HDB solution containing collagenase II (0·04 %; Worthington Biochemical Corporation, Freehold, NJ, USA) and deoxyribonuclease I (Sigma) as previously described (Fora et al. 1996). Briefly, polypropylene centrifuge tubes, containing the tissue fragments and digestion solution, were placed on an orbital shaker and gently rocked for 2·5 h at 37°C. The AP fragments were then mechanically triturated and centrifuged for 5 min at 400 g. The cell suspension was then washed in 7·5 ml complete medium (Dulbecco's Modified Eagles Medium (DMEM) plus Ham's F12 medium (1 : 1) and charcoal-stripped fetal calf serum and heat-inactivated horse serum both added to a final concentration of 10 %) and centrifuged. The cell suspension was then washed and centrifuged again. Cells were plated in complete medium, at (2·0-3·0) × 105 cells per well in 48-well tissue culture plates (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ, USA) in a 1 ml volume. Cells were cultured at 37°C in a water-saturated 5 % CO2 atmosphere (BB 16 gas incubator; Haraeus Instruments, GmbH, Hanau, Germany).
Experimental protocol
The cells of a single fetal sheep pituitary were used for each culture, i.e. pituitary glands were not pooled for experiments. Individual experiments (i.e. exposure of cells from one pituitary to vehicle alone, CRH, AVP or CRH + AVP) involved the measurement of ACTH responses in replicate wells for each experiment and these were then averaged to yield one datum point. The number of experiments cited (n) therefore reflects the total number of pituitary glands used in each case. Twenty-four hours after plating, the cells in half the wells from each pituitary were treated with Cx at a final concentration of 25 nmol l-1. Cx was synthesized as previously described (conjugate of Nle21,38,Arg36 rat CRH and the toxin gelonin) (Schwartz et al. 1987). Complete medium (vehicle) was added to the cells in the remaining wells. After 18 h exposure to Cx or vehicle, cells were washed with complete medium and returned to culture. After a further 48 h, the cells were washed 3 times with incubation medium (DMEM plus F12 (1 : 1) 0·2 % Polypep) and allowed to equilibrate in serum-free conditions for 1 h. Cells were then washed in incubation medium and treated for 3 h with either medium alone (control; n = 31), ovine (o)CRH (10-8 M; n = 16), AVP (10-7 M; n = 20) or oCRH + AVP (n = 18). The concentrations of CRH and AVP used in these experiments were selected on the basis of previous in vitro studies with adult sheep pituitary cells and in vivo measurements of CRH and AVP concentrations in portal blood (Engler et al. 1989; Kemppainen et al. 1993). After 3 h the culture medium was collected and stored at -20°C and the cells were stored in 0·1 M HCl (1 ml) at -80°C for ACTH radioimmunoassay. Cellular extracts of ACTH were obtained by thawing and refreezing the cells to -80°C 3 times and using mechanical trituration on either the second or third thaw. The extracts were stored at -20°C.
ACTH radioimmunoassay
The concentration of immunoreactive (ir)ACTH in the cell culture medium and in the cellular extracts was measured using a double antibody radioimmunoassay as previously described (McMillen et al. 1990), validated for use in cell culture media. Synthetic human ACTH1-39 (Peninsula Laboratories, Belmont, CA, USA) was used as the standard (1·95-500 pg tube-1). Anti-ACTH antiserum (Anti-ACTH 1000T; ICN Biomedicals, Inc., Costa Mesa, CA, USA) was raised in rabbits against a purified porcine ACTH conjugate. The anti-ACTH cross-reacted 100 % with ACTH1-39 and ACTH1-24 and < 0·1 % with 
-melanocyte-stimulating hormone (MSH) and 
MSH (information supplied by ICN Biochemicals Inc.). The sensitivity of the assay was 1·95 pg tube-1. Human ACTH1-39 (7·8-31·2 pg tube-1) added to the cell culture medium was quantitatively recovered (109 ± 5 %; n = 9). The intra- and interassay coefficients of variation were less than 10 %.
Cortisol radioimmunoassay
Cortisol was measured after extraction from plasma with dichloromethane (2 ml, AnalaR, Merck Pty Ltd, Kilsyth, Victoria, Australia) using the Orion Diagnostica cortisol kit (Orion Diagnostica, Espoo, Finland) as previously described (Bocking et al. 1986). The efficiency of the recovery of cortisol from fetal sheep plasma was 84 ± 1 %.
Statistics
All values are expressed as means ± standard error of the mean (S.E.M.). The total amount of ACTH stored and secreted in cells in each well during basal, i.e. non-stimulated, conditions was calculated for each pituitary in the three treatment groups (116 days gestation, saline infused (116d + Sal); 116 days gestation, cortisol infused (116d + F); and 140-145 days gestation (140-145d)) with or without pretreatment with Cx. The effects of Cx on the amount of ACTH stored and secreted by corticotrophic cells in the three treatment groups were then compared using analysis of variance (ANOVA) with group and Cx pretreatment as the major factors.
The proportion (%) of total ACTH (ACTH cell content + secreted ACTH) present in CRH-responsive cells (i.e. Cx-sensitive cells) was calculated as the ratio of (total ACTH content - ACTH content after Cx pretreatment) : total ACTH content and was compared in the three treatment groups using one-way ANOVA. The effect of Cx on the amount of ACTH secreted (expressed as a percentage of the total ACTH available for secretion) was also compared in the three treatment groups using multifactorial ANOVA. The ACTH secretory responses to CRH (10-8 M), AVP (10-7 M) and CRH + AVP were compared in the three treatment groups with or without Cx pretreatment using multifactorial ANOVA. Duncan's post hoc test was used to identify significant differences between mean values after ANOVA and a probability of 5 %, i.e. P < 0·05 was taken to be significant.
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RESULTS |
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Impact of age, cortisol and CRH-cytotoxin on ACTH content and secretion
The mean fetal plasma concentration of cortisol was higher (P < 0·05) in the 116d + F group (46·8 ± 4·4 nmol l-1) than in the 116d + Sal group (1·83 ± 0·15 nmol l-1).
There was no significant difference in the total ACTH (ACTH cell content + secreted ACTH) present in the three treatment groups (116d + Sal, 618 ± 198 pg ACTH (104 cells)-1; 116d + F, 464 ± 82 pg ACTH (104 cells)-1; 140-145d, 781 ± 238 pg ACTH (104 cells)-1) or in the proportion of total ACTH secreted during the 3 h control period (116d + Sal, 1·16 ± 0·23 %; 116d + F, 2·45 ± 0·95 %; 140-145d, 1·73 ± 0·25 %) (Fig. 1).
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The proportion (means + S.E.M.) of total ACTH secreted during basal conditions in the three treatment groups (116d + Sal, n = 10; 116d + F, n = 11; 140-145d, n = 10) in the absence ( | ||
Pretreatment with Cx resulted in a significant decrease (F = 142·8, P < 0·001) in the total amount of ACTH present in the 116d + Sal group (no Cx, 16·8 ± 5·5 ng ACTH well-1; Cx, 6·5 ± 3·1 ng ACTH well-1) and the 140-145d group (no Cx, 22·9 ± 7·7 ng ACTH well-1; Cx, 7·1 ± 3·1 ng ACTH well-1). The effect of Cx on total ACTH was significantly greater (P < 0·05) in these groups, however, than in the 116d group which had been infused with cortisol in vivo (116d + F; no Cx, 12·6 ± 4·0 ng ACTH well-1; Cx, 8·2 ± 3·3 ng ACTH well-1).
There was no significant difference in the proportion of total ACTH which was present in Cx-sensitive cells between the 116d + Sal (71 ± 4·5 %) and 140-145d (71 ± 4·4 %) groups. After cortisol infusion in vivo, however, there was a significant reduction (F = 6·42, P < 0·01) in the proportion of total ACTH which was present in the Cx-sensitive cells (46 ± 7·5 %) during basal conditions compared with either the 116d + Sal or the 140-145d groups (Fig. 2).
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The proportion (means + S.E.M.) of ACTH present in CRH-responsive cells in each of the three treatment groups. The asterisk indicates that the proportion of ACTH present in the CRH-responsive cells was significantly lower in the 116d + F group (n = 11) than in either the 116d + Sal (n = 10) or 140-145d (n = 10) groups. | ||
After Cx pretreatment, there was a similar significant increase (F = 65·4, P < 0·001) in the proportion of total ACTH which was secreted in the 3 h control period in all three treatment groups (116d + Sal, 6·30 ± 1·49 %; 116d + F, 4·05 ± 1·33 %; 140-145d, 5·80 ± 1·67 %) (Fig. 1).
Effect of Cx on ACTH secretory responses to CRH and AVP
Gestational age or cortisol infusion in vivo had no effect on the ACTH secretory responses to the hypothalamic secretagogues.
There was a significant interaction (P < 0·001) between the effects of Cx and the effects of the secretagogues on the ACTH secretory response. In the absence of Cx pretreatment, the proportion of total ACTH secreted was significantly higher (P < 0·05) in response to CRH + AVP in all three groups than in response to either CRH or AVP alone or to vehicle (Table 1). The proportion of ACTH secreted in response to AVP was also significantly higher (P < 0·05) than in response to CRH (10-8 M) or to vehicle (Table 1).
Table 1. The percentage of total ACTH secreted from pituitary cells in response to CRH, AVP, CRH + AVP or vehicle with or without pretreatment with Cx
| No Cx pretreatment | Cx pretreatment | |||||
| 116d + Sal | 116d + F | 140-145d | 116d + Sal | 116d + F | 140-145d | |
| CRH + AVP | 9·8 ± 2·7 d | 11·5 ± 4·7 d | 14·7 ± 3·9 d | 22·5 ± 3·2 b | 17·4 ± 4·5 b | 18·8 ± 2·9 b |
| AVP | 5·0 ± 1·6 c | 4·0 ± 1·1 c | 5·5 ± 1·5 c | 21·1 ± 4·3 b | 11·5 ± 1·5 b | 17·8 ± 2·7 b |
| CRH | 3·5 ± 1·8 b | 2·8 ± 1·1 b | 2·1 ± 0·9 b | 7·7 ± 3·4 a | 4·6 ± 1·3 a | 4·4 ± 1·0 a |
| Vehicle | 1·2 ± 0·2 a | 2·5 ± 1·0 a | 1·7 ± 0·3 a | 6·3 ± 1·5 a | 4·0 ± 1·3 a | 5·8 ± 1·6 a |
After pretreatment with Cx, the proportion of total ACTH secreted in response to CRH was not significantly greater than the ACTH response to vehicle. Furthermore, the ACTH secretory responses to CRH + AVP and to AVP were the same and significantly greater than the responses to either dose of CRH or to vehicle (Table 1).
There was also no significant difference in the ACTH responses to AVP (expressed as x-fold change from baseline) between cells not exposed or exposed to Cx pretreatment (116d + Sal, 5·5 ± 1·7 vs. 5·8 ± 1·3, respectively; 116d + F, 4·6 ± 1·9 vs. 7·7 ± 3·1, respectively; 140-145d, 3·6 ± 1·0 vs. 4·9 ± 1·5, respectively).
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DISCUSSION |
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There was no significant change in the total amount of ACTH present in the fetal pituitary cells or in the proportion of ACTH which was secreted under basal conditions between 116 and 145 days gestation. The increase in plasma ACTH concentration which occurs during the last 15-20 days of gestation in the fetal sheep (Challis & Brooks, 1989; Ozolins et al. 1991) is dependent on a functional fetal hypothalamus (Phillips et al. 1996), and it is possible that the stimulatory effects of the hypothalamic drive may not persist in fetal pituitary cells after 4-5 days in culture. It is also not clear whether there is an increase in the ACTH synthetic capacity of all corticotrophic cells in the fetal pituitary before birth. There is a differential level of expression of the ACTH precursor pro-opiomelanocortin (POMC) in the separate regions of the anterior lobe of the fetal sheep pituitary with highest expression in the basal or inferior portion of the pars distalis (Matthews et al. 1994) and there are conflicting reports of an increase (Yang et al. 1991; Myers et al. 1993; Matthews et al. 1994) and a decrease (McMillen et al. 1988; Brooks et al. 1992; Merei et al. 1993) in POMC mRNA levels in the anterior pituitary of the fetal sheep in the weeks before delivery.
Pretreatment with the CRH-toxin conjugate resulted in a consistent increase in the proportion of ACTH secreted in vitro. This was a consequence of maintained ACTH secretion in the face of a decrease in cellular ACTH content. It has previously been reported that pretreatment of adult sheep pituitary cells with the CRH-cytotoxin also increased the basal levels of POMC mRNA and ACTH secretion (van de Pavert et al. 1997). Furthermore, after elimination of the CRH target cells in the adult pituitary, the remaining AVP-responsive cells secrete the same or more ACTH as in the intact population, although fewer corticotrophs are present (van de Pavert et al. 1997). Our data also suggest that under basal or unstimulated conditions, ACTH may be secreted primarily from a subpopulation of corticotrophs in the fetal pituitary which are not CRH responsive (i.e. the AVP-responsive corticotrophs).
CRH-responsive corticotrophs
Whilst the basal level of ACTH secretion is maintained, there is a significant decrease in pituitary ACTH content after CRH-cytotoxin pretreatment, consistent with the selective removal of a population of cells which are CRH responsive. It is particularly interesting that the same proportion (approximately 70 %) of pituitary ACTH was present in CRH-responsive corticotrophs at 116 days and after 140 days gestation. Whilst the fetal sheep in the early gestational age group were exposed to fetal surgery and catheterization at around 103 days gestation, it is unlikely that there were any long-lasting effects of this surgery that impacted on the fetal corticotrophs, given the low circulating levels of fetal cortisol in this group at 116 days gestation. There are significant changes in the morphology of corticotrophic cells in the fetal sheep pituitary across this gestational age range (Perry et al. 1985; Mulvogue et al. 1986; Antolovich et al. 1989). The main corticotroph observed between 90 and 130 days gestation is a tall, columnar cell or 'fetal' corticotroph these cells are arranged in large clumps or palisades. After 135 days gestation the predominant corticotroph in the fetal pituitary is a small angular and intensely ACTH immunoreactive 'adult' cell type (Perry et al. 1985; Mulvogue et al. 1986). Our data indicate that despite the change in the predominance of the fetal cell type in mid-gestation to the adult cell type in late gestation, there is no substantial change in the proportion of ACTH stored in corticotrophs which are CRH responsive. This suggests that there is not a direct relationship between the predominance of the morphologically distinct corticotrophs and the proportion of ACTH present in CRH-responsive corticotrophs in the fetal sheep pituitary. It is intriguing that most of the ACTH in the fetal pituitary is present in cells which are CRH responsive.
There is evidence from studies in the adult sheep that in CRH-responsive cells, the classically regulated protein secretory pathway operates, which allows for greater processing of POMC to ACTH1-39. Furthermore, there is evidence that AVP-responsive corticotrophs appear to synthesize and secrete ACTH via an alternately regulated pathway which results in less opportunity for processing of the ACTH precursors (Schwartz et al. 1991). There is evidence from in vitro and in vivo studies, using separate immunoradiometric assays specific for ACTH1-39 and the ACTH precursors, that there is an increase in the pituitary secretion of ACTH1-39 relative to the ACTH precursors in late gestation (Carr et al. 1995; McMillen et al. 1995; Phillips et al. 1996). Given that most of the ACTH appears to be within the CRH-responsive corticotrophs throughout late gestation, the change in the output of ACTH1-39 after 140 days gestation, may be due either to maturation of post-translational processing events within these corticotrophs or to increased hypothalamic stimulation of these cell types in late gestation.
Infusion of cortisol into the fetus for 7 days significantly decreased the proportion of ACTH present in CRH-responsive cells in the fetal pituitary. Matthews & Challis (1997) have reported that whilst cortisol decreased both CRH- and AVP-stimulated ACTH secretion in fetal pituitary cells in culture, this effect was greater after CRH than AVP. It has been previously shown in intact populations of pituitary cells from adult sheep that pretreatment with dexamethasone for 16-18 h decreased the ACTH secretory responses to AVP or CRH in intact populations of sheep pituitary cells in culture, but did not decrease the residual ACTH response to AVP in populations previously treated with CRH-cytotoxin (Schwartz et al. 1994). Neill and coworkers (1987) also concluded from studies with the reverse haemolytic plaque assay that there are two functional subpopulations of corticotrophs, one of which is differentially responsive to CRH and preferentially inhibited by glucocorticoids. We can also conclude from the present study, that cortisol acts preferentially to inhibit ACTH synthesis in fetal pituitary corticotrophic cells which are CRH responsive. We have previously found in perifusion studies, that cortisol acts in vitro in a short time domain to suppress the secretion of ACTH1-39 but not the ACTH precursors in late gestation (McMillen et al. 1995). This provides further evidence that cortisol acts primarily on corticotrophic cell types which secrete relatively more ACTH1-39. Whilst cortisol decreased the proportion of ACTH present in the CRH-responsive cells, it had no effect on the amount of ACTH present in the Cx-resistant cells. We conclude therefore that the AVP-responsive cells in the fetal pituitary are relatively resistant to the negative actions of cortisol on ACTH synthesis and secretion.
In the present study, whilst we achieved fetal cortisol concentrations at 116 days gestation similar to those present after 140 days gestation, the proportion of ACTH in the CRH-responsive corticotrophs in the cortisol-treated group was significantly lower than that after 140 days gestation. It may be that after 140 days gestation, the stimulatory influence of the fetal hypothalamus counteracts the negative feedback effect of cortisol on ACTH synthesis in the CRH-responsive cells. Electrolytic lesions of the paraventricular nuclei or surgical disconnection of the fetal hypothalamus and pituitary each prevent the normal prepartum increase in ACTH and delay parturition (McDonald & Nathanielsz, 1991; Phillips et al. 1996).
ACTH responses to CRH and AVP
In the present study, we found that the ACTH secretory responses to AVP and to CRH + AVP were significantly greater than the responses to CRH throughout late gestation and after cortisol infusion into the fetus. Previous studies have also reported that the ACTH secretory responses to CRH + AVP and to AVP were greater than to CRH after 138 days gestation (Fora et al. 1996). The ACTH secretory response after CRH, in these studies, however, was reported to be greater than after AVP at around 108 days gestation (Fora et al. 1996). In the present study whilst we found a higher ACTH response to AVP than CRH independent of gestational age, there was a trend towards a greater ACTH response to CRH in early gestation and the inclusion of all variables (age, Cx pretreatment, secretagogues) in the multifactorial ANOVA may have limited the capacity to define gestational trends in the ACTH secretory responses to CRH. Perez and coworkers have recently used immunoblotting techniques to study the effects of CRH and AVP on the types of ACTH secretory responses in fetal sheep corticotrophs (Perez et al. 1997). At both 120 and 135 days gestation, AVP, alone or in combination with CRH, increased the proportion of secreting corticotrophs by about 60 %. At 135 days gestation, AVP or AVP + CRH treatment also increased the amount of ACTH secreted by each cell. In contrast, at 120 days, CRH stimulated a subpopulation of corticotrophs to release ACTH, but at around 135 days, the ACTH response to CRH had changed to one that involved an increased output from cells which were already secreting ACTH. The authors postulated that a number of changes, including an increase in AVP receptor expression (Shen et al. 1990) and a decrease in CRH receptor expression (Lu et al. 1991) may explain the changes in pituitary responsiveness to the hypothalamic secretagogues.
In the present study, it was particularly interesting that the ACTH response to AVP remained constant at around 4- to 8-fold above basal output even after Cx pretreatment, i.e. after the elimination of CRH-responsive cells. One explanation for this result is that, in the fetal sheep pituitary, the majority of corticotrophs which are AVP responsive are responsive to AVP only, rather than to CRH and AVP.
Summary
In summary, therefore, we have clearly defined separate subpopulations of corticotrophs in the anterior pituitary of the fetal sheep which are CRH and AVP responsive. We have demonstrated that around 70 % of ACTH in the fetal anterior pituitary is stored within corticotrophs which are CRH responsive. We have also found that whilst there was no change in this pattern with increasing gestation, cortisol acts preferentially to inhibit ACTH synthesis in fetal pituitary corticotrophic cells which are CRH responsive and that the AVP-responsive cells are relatively resistant to the negative effects of cortisol. Our data suggest that the stimulatory influence of the fetal hypothalamus must counteract the negative feedback effect of cortisol on ACTH synthesis in the CRH-responsive cells to stimulate the increase in pituitary ACTH1-39 output which occurs before delivery.
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The authors gratefully acknowledge financial support from the National Health and Medical Research Council of Australia for these studies. The authors also acknowledge the expert surgical and technical assistance of Giuseppe Simonetta, Simon Fielke, Frank Carbone and Anne Jurisevic in the conduct of these studies.
Corresponding author
I. C. McMillen: Department of Physiology, The University of Adelaide, Adelaide, SA 5005, Australia.
Email: cmcmillen{at}physiol.adelaide.edu.au
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) and presence (
) of the CRH-cytotoxin conjugate. The asterisks indicate significant differences between the percentage of ACTH secreted in the absence and presence of Cx in each treatment group.