Int. J. Biochem. Vol. 24, No. 1,pp.79-84, 1992 Printed in Great Britain.

0020-711X/92$5.00+ 0.00 Pergamon Press plc

ONTOGENETIC CHANGES IN PORCINE ADRENOCORTICAL ADRENOCORTICOTROPIC HORMONE RECEPTORS* HAROLD G. KLEMCKE~ U.S. Department of Agriculture, Agricultural Research Service, Roman L. Hruska U.S. Meat Animal Research Center, Clay Center, NE 68933-0166, U.S.A. [Tel. (402) 762-4189; Fax (402) 762-41481 (Received

26 February

1991)

Abstract-l. Binding of [12sI]ACTH(I-38) analog to adrenal receptors was measured in fetal pigs (Sus domesticus) at 15day intervals from midpregnancy (60 days) to near term (105 days; pregnancy length 114 days). 2. Binding was greatest at day 60 (0.42 + 0.03 fmo1/200 pg protein or 0.50 _+0.08 fmol/50 pg DNA), and least at day 105 (0.13 f 0.03 fmo1/2OOpg protein or 0.16 + 0.04 fmol/50 pg DNA). Total adrenal binding was constant (0.61 + 0.02 fmolipaired adrenals). 3. Scatchard analyses at day 60 and day 105 showed comparable apparent affinities of ACTH receptors (K, day 60 = 1.51 + 0.72 x 109M-r vs K, day 105 = 1.94 f 0.78 x 109M-‘). 4. DNA per paired adrenals and membrane-associated protein increased l.dfold, providing a constant protein: DNA ratio. Concentrations of adrenal cortisol were constant from 60 to 90 days of gestation age but increased dramatically by day 105. 5. These data suggest that during 60-105 days of gestation age the number of ACTH receptors per cell is reduced.

of the biologically active monoiodo-Tyr23,Phe2,Nle4ACTH(l-38) analog (Buckley et al., 1981). A knowledge of changes in adrenal ACTH receptors during fetal growth would not only provide information concerned with fetal adrenal development, but also could indicate potential developmental changes in responsiveness of the fetal adrenal to ACTH.

INTRODUCTION Fetal adrenal development is dependent on the presence of the fetal pituitary gland in all species studied, including pigs (Jost, 1966; Bose et al., 1974;

Jaffe, 1982). Adrenocorticotropic hormone (ACTH) is one fetal pituitary factor responsible for fetal adrenal development (van Dijk and Challis, 1989; Pepe and Albrecht, 1990). Fetal adrenal function in turn promotes functional and morphological development of many tissues that include the liver, pancreas, lung, and brain (Liggins, 1976). In order for the fetal adrenal to respond to ACTH, it must first develop ACTH receptors. Detailed ontogenetic studies of adrenal ACTH receptors have only been conducted with sheep (Durand, 1979) and rats (Chatelain et al., 1989) and were made during late gestation. Very limited measurements of human fetal adrenal ACTH receptors were made at midgestation (Jaffe, 1982). Adrenal development in fetal pigs has been largely overlooked, and the few studies conducted have been concerned with the periparturient period (Dvorak, 1972; Brenner et al., 1978; Lohse and First, 1981; Randall, 1983). The current studies were conducted, therefore, to measure changes in adrenal size, cortisol content, and ACTH receptors from near midpregnancy to near term (60-105 days of a 11Cday gestation period). In these studies, use was made

MATERIALS

AND METHODS

Animals and tissue collections Sows of the Duroc lean x Yorkshire lean cross that were bred and raised in the USDA-ARS Roman L. Hruska U.S. Meat Animal Research Center (MARC) swine facilities were used in this study. These pigs were housed in an indoor confinement facility with a photoperiod consisting of 14 hr light per day, and an environmental temperature of 15.518°C. Water was provided ad libitum, and sows were fed 1.6 kg of meal consisting of 13% crude protein during O-80 days of gestation, and 2.5 kg thereafter. Reproductive tracts were removed at slaughter. Uterine horns were dissected to expose the fetuses that were then removed, and subsequently adrenal glands were dissected from the fetuses. In this study, sows were slaughtered at 60, 75, 90, and 105 days of pregnancy (average length of pregnancy in pigs is 114 days). Adrenal glands were obtained from a total of 190 fetuses, and were combined within each gestational age and across sexes (in approximately equal numbers) to provide sufficient tissue for the ACTH receptor assay, and according to the following scheme: day 60, two tissue pools, 55 fetuses; day 75, three tissue pools, 46 fetuses; day 90, five tissue pools, 51 fetuses; day 105, six tissue pools, 38 fetuses. All procedures were in compliance with the MARC Animal Care Guidelines. Once adrenal glands were removed from the animals, they were placed in a 0.01 M sodium phosphate buffered saline (0.15 M) solution, pH 7.4. Subsequently, adipose tissue was dissected from the adrenals that were then weighed, placed

*Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may also be suitable. tPlease address all correspondence to: Harold G. Klemcke, USDA-ARS, U.S. Meat Animal Research Center, P.O. Box 166, Clay Center, NE 68933-0166, U.S.A. 79

HAROLD G. KLEMCKE

80

in 12 x 75 mm polypropylene tubes, rapidly frozen in liquid nitrogen, and stored frozen at -80°C. Preparation of tissue for receptor assay

Adrenal glands were thawed, decapsulated, and then minced and placed in 4 ml of ice-cold 0.3 M sucrose, 0.025 M Tris-HCl buffer solution @H 7.5 at 4X), which also contained 0.01 M CaCl,, and 0.1% sodium azide. The tissue was then homogenized on ice for 1 min with a Polytron PTlO (Brinkman Instruments, Westbury, N.Y.). This homogenate was initially filtered through the stainless steel support screen of a Gehnan Easy-Pressure syringe filter holder (No. 4320. Ann Arbor. Mich.). and the filtrate centrifuged at 149,&O gav for 1 hr at 2”C:‘Subsequently, the pellet was washed three times by addition and subsequent removal of 1 ml of 0.025 M Tris-HCl (pH 7.5 at 25°C) containing 0.01 M CaCl, and 0.01% sodium azide (hereafter called ACTH-I), rehomogenized in 4 ml of ACTH-1, and recentrifuged at 149,OOOg,,for 1 hr at 2°C. The supernatant was then aspirated, the pellet weight determined, and the pellet resuspended via homogenization (as above-noted) at a concentration of 20-40 mg/ml of ACTH-1. ACTH

receptor assay

Tissue preparation, iodination, hormone purification, and receptor assay procedures have been described in detail elsewhere (Klemcke and Pond, 1991) and are essentially those of Buckley and co-workers (Buckley and Ramachandran, 1981; Buckley et al., 1981). In brief, Phe2,Nle4-ACTH(l-38) analog was iodinated using a chloramine-T procedure; purified initially on a Sephadex LH-20 column (Phannacia Inc., Piscataway, N.J.) and the eluent containing the iodinated hormone stored for up to 2 weeks at 2-4°C. Prior to use, the iodinated hormone was repurified by high performance liquid chromatography (HPLC) using an ultrasphere octadecylsilane (ODS) 5 pm, reversed-phase HPLC column (Beckman Instruments Co., Altex Division, San Ramon, Calif.). Isocratic elutions were conducted using a mobile phase that consisted of 1.0 M pyridine, 0.51 M acetic acid, and 14% I-propanol. Fractions containing the monoiodinated ACTH analog were pooled, and the labelled hormone exchanged into an aqueous buffer. The specific activity of labelled ACTH preparations-experimentally determined in two preparations by self-displacement in a radioligand receptor assay (Catt et al., 1976)-was 2674 f. 220 Ci/mmol (mean f SEM), which (based on the specific activity of the ‘*‘I used and correcting for decay) computed to an average of 1.21 atoms of iodine per molecule ACTH analog. Assays were conducted in 12 x 75 mm polypropylene disposable test tubes and in a final incubation volume of 500 ~1. The incubate included 100 pl of monoiodinated ACTH analog, 100 ~1 of receptor preparation, and 300 ~1 of ACTH-1 containing 1% bovine serum albumin (BSA, hereafter called ACTH-2). For all tissue pools, saturation analyses were attempted by incubating receptor preparations with a single nonsaturating concentration of [iZSBACTH analog (250,000 dpm; 84 p-M), and with seven seriai dilutions of u&belled AC?TH analog (49-3 131 uM). However. technical difficulties with many-of these serial’dilutions precluded their use. Hence, data are presented for the single nonsaturating concentration of labelled ACTH analog, and for usable Scatchard analyses that were obtained at 60 and 105 days of age. There were four assays conducted for this study. The within-assay coefficient of variability (CV) based on triplicate determinations of binding of the nonsaturating concentration of [12SI]ACTHfor the 16 tissue pools was 7.2%. The betweenassay CV based on a receptor preparation that was assayed in all four assays was 4.4%. Nonspecific binding was also measured in triplicate for each experimental sample in the presence of 50 ng cortrosyn (34 nM). Incubation-associated degradation of ACTH analog was measured using a trichloroacetic acid (TCA) precipitation procedure (Klemcke

and Pond, 1991). In this procedure, after the initial incubation for 40min and incubation termination, a 50 ~1 aliquot of supematant was removed and added to 1 ml of ice-cold 10% TCA, mixed thoroughly, and incubated on ice for 15 min. These incubates were subsequently centrifuged at 8OOOg_ for 10min at 2”C, and the supematant aspirated. An additional 1 ml of ice-cold TCA was added, tubes mixed thoroughly, and then immediately recentrifuged at 8OOOg,, for 10min at 2°C. The supematants were again aspirated, and radioactivity in pellets was determined. Cortisol radioimmunoassay Cortisol in fetal adrenal glands was measured using a cortisol radioimmunoassay kit supplied by Diagnostic Products (Los Angeles, Calif.). Fetal adrenal glands were processed as above-noted for the ACTH receptor preparation, and the first 149,OOOg,, centrifugation supematant was used for cortisol analysis. No measure of potential losses of cortisol during homogenization and filtration was made. Such losses were assumed to be randomly distributed across all tissue pools. The sensitivity of this assay as measured by the lowest standard in the linear range of the standard curve was 156pg/tube. Serial dilutions of supernatants from two fetal adrenal tissue pools had slopes (- 1.01 and -0.99) that did not differ (P > 0.05) from that of the standard curve (-0.95). Accuracy of the assay as measured by addition of four quantities of cortisol (0.32.5 ng) to a fetal adrenal 149,OOOg,,supematant was 104%. The precision of the assay as measured by the average CV of the 16 tissue preparation supematants analyzed in duplicate was 2.5%. Protein assay

Protein content of receptor samples was measured in triplicate using a modification of the Lowry procedure (Markwell et al., 1978), which was previously validated for use with receptor membrane preparations (Klemcke et al., 1981). DNA assay

DNA measurements were conducted using a fluorometric procedure detailed by LaBarca and Paigen (1980). Calf thymus DNA was used as a standard. This assay had a sensitivity of 0.16pg DNA. Accuracy of estimates was determined by addition in quadruplicate of three quantities of DNA (0.63-2.5 fig) to aliquots of a receptor preparation. The average accuracy of estimates was 107% with a withinassay CV of 1.98%. Experimental design and statistics

Data were initially analyzed by analysis of variance to test for main effects and interactions, and the StudentNewman-Keul’s test was used to test for significant differences among specific means. All data were examined for homogeneity of variance using the F_ test (Snedecor and Cochran, 1967) and for normality of distribution via the Shapiro Wilk Statistic. Data were log or square root transformed where necessary to fulfill assumptions of analysis of variance. A probability level of co.05 was considered significant. Scatchard analyses were conducted using the computer program LIGAND (Munson and Rodbard, 1980), which makes use of weighted nonlinear least-squares analysis. In these analyses nonspecific binding was not subtracted from the data. Instead, the LIGAND subroutine SCAFIT was used to compute the nonspecific binding as a data-determined parameter (Munson and Rodbard, 1980; Mendel and Mendel, 1985). Comparison among apparent equilibrium association constants (JQ were also conducted using procedures associated with the LIGAND program. In these procedures, the slope of each individual Scatchard plot is compared with that of every other Scatchard (A vs B, B vs A, A vs C, C vs A, etc.). For the comparison of slope A with slope B to be considered significant, both A vs B and B vs A must be significant.

Fetal porcine adrenal ACTH receptors RESULTS

As gestation progressed from near midpregnancy to almost term, fetal weights increased I-fold whereas paired adrenal weights increased 3-fold (Table 1). There were no differences between male and female adrenal weights at any gestational age (data not shown). The ratio of paired adrenal weights to body weight was greatest at 60 days of age and progressively declined thereafter (P < 0.05). Concentrations of DNA and protein remained constant throughout the gestational ages measured (Table 2), and total quantities per paired adrenal glands increased (P < 0.05). The ratio of total particulate protein to total DNA was constant. As adrenal glands enlarged, binding to a single, nonsaturating concentration of [‘*‘I]ACTH analog was reduced [P < 0.05; Fig. l(A)] when expressed on either a per 200 pg protein or on a per 50 pg DNA basis. Total binding (fmol/paired

81

adrenals) of a single concentration of [‘*rI]ACTH analog to adrenal ACTH receptors was essentially unchanged [P -z 0.05; Fig. l(B)] as adrenal glands grew between 60 and 105 days of gestational age. Data from the two tissue pools at day 60, and four pools at day 105 indicated K, of 1.51 k 0.72 x Table

1.Porcine fetal measures at different gestational ages

Gestational a8= Ways) n 60 75 90 105

Fetal weight (8).

Ratio Paired adrenal weight adrenal weight to body weight (md*

103.1 * 1.7’ 263.7 & 5.1b 514.8 f 9.6’ 942.1 f 30.3d

55 46 51 38

25.9 k 0.9* 35.1 * 1.4b 62.4 f 1.8= 97.9 +_4.2d

0.246 * 0.137 * 0.122 f 0.105 +

0.007’ 0.006b 0.003c 0.004d

*Body weights and weights of adrenal glands are for male and female adrenal glands combined. Data represent the mean k SEM. Means with different letter superscripts for a given measure are significantly different (P c 0.05).

Table 2. Porcine fetal adrenal-associated measures Gestational age (days)

n

60 75 90 105

(2) (3) (5) (6)

DNA Us) mg tissue 17.47 f 18.19 f 15.91 * 15.07 *

2.28” 0.65’ 1.11’ 1.22a

81.0 k 127.7 f 130.4 * 212.7 f

Protein @g)* Paired adrenals

Protein @g)* mg tissue

DNA ol8) Paired adrenals 15.0’ 6.7’b lO.Wb 27.9b

82.5 + 77.47 + 67.80 f 69.5 k

381.3 k 542.9 f 554.5 f 982.9f

3.50’ 3.27’ 6.19 4.41’

30.0a 10.2’ 49.5a 118.1b

Ratio Total Protein Total DNA 4.79 f 4.28 k 4.25 + 4.66 f

0.44” 0.24” 0.20’ 0.21’

*Membrane associated protein. Each datum point represents the mean * SEM of the number of tissue pools indicated in parentheses. Means with different superscripts for a given measure are different (P < 0.05).

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Fig. 1. (A) Specific binding of labelled ACTH analog to porcine adrenal ACTH receptors at various gestational ages when expressed on a concentration basis. (B) Total specific binding of labelled ACTH analog to porcine adrenal ACTH receptors at various gestational ages. (C) Incubation-associated degradation of lab&xl ACTH analog measured by its precipitability in trichloroacetic acid (TCA) and after correcting for labelled analog which is undamaged and not precipitable by TCA. (D) Adrenal cortisol in porcine adrenal glands at various gestational ages. ac

2411-F

82

HAROLD G. KLEMCKE

Fig. 2. Scatchard analyses of average binding to fetal adrenal ACTH receptors using least-squares linear regression analysis of data with nonspecific binding subtracted. Tabular inset reflects the 2 f SEM of individual final Scatchard analyses using computer program LIGAND without subtracting nonspecific binding, hence there are differences between graphical and tabular data. For day 60 graph, n = 2 fetal adrenal pools; for day 105, n = 4 fetal adrenal pools.

lo9 M-’ (B,,,, = 28.34 f 13.84 fmol/mg

protein) and 1.94 + 0.78 x lo9 M-’ (B,,,,, = 14.78 f 8.79 fmol/mg protein), respectively (Fig. 2). One of these latter four tissue pools from day 105 had a very high concentration of ACTH receptors and in its absence the average B,,,, at day 105 was 6.26 + 3.0fmol/mg

protein. Formation of hormone-receptor complexes (if they obey the laws of mass action) should depend both on the number of receptors present, and on the affinity of the receptors for the ligand. Indeed, binding at the single concentration of labelled ligand was not correlated with either B,,,,, or K, individually in those tissue pools for which Scatchard analyses were obtained (P > 0.05). However, this binding was highly correlated (r = 0.978) with the product of B,,, and K, (P < 0.01). There were also receptor preparation-associated changes in [‘*‘I]ACTH degradation. The amount of degradation activity occurring in the tubes (which reflects both the degradation activity of that preparation and the amount of preparation present) decreased approximately 6% (P < 0.001) as fetal age increased [Fig. l(C)]. When normalized to a constant amount of protein, the degradative activity increased modestly from 60 to 90 days of age (P > 0.05), then decreased dramatically by day 105 (P < 0.05). Fetal adrenal concentrations of cortisol were stable during days 60-90 of gestation, but dramatically increased (with total content of cortisol) by day 105 or approximately 9 days prior to parturition [Fig. l(D)]. DISCUSSION

The data presented herein represent the first for swine that measure fetal changes in adrenal ACTH receptors. The current data also extend those obtained for sheep and rats (Durand, 1979; Chatelain et al., 1989) since they provide information for an earlier gestational period, and measure binding to an ACTH receptor with an apparent affinity closely approximating that needed to be of physiological relevance. The data suggest an increased amount of binding of ACTH to its adrenal receptors at day 60

of gestational age when compared with later gestational ages. The Scatchard analyses indicate that the increased binding reflects an increase in number, not affinity, of receptors. However, the low number of tissue pools in day 60 necessitates tempering the strength of conclusions made. As the adrenal glands grew in size, the quantity of DNA and protein per paired adrenals increased proportionately. This increased DNA and constant protein/DNA ratio suggests that this growth was predominantly due to hyperplasia. Previous histological observations indicated that both hypertrophy and hyperplasia of porcine adrenocortical cells occurred between 89 and 105 days of gestational age with hyperplasia predominating between days 105 and 113 (Lohse and First, 1981). Hence, the data suggest that as the adrenal glands grow via predominantly hyperplasia between days 60 and 75, there is a decrease in the number of ACTH receptors per cell. Additional supportive evidence for this conclusion occurs by comparing ACTH adrenal binding on day 105 with that on day 60. Not only is there a reduced binding per mass of DNA, but also total binding in the adrenal glands remained constant concomitant with an incease in number of cells (pg DNA). Since the percent degradation of labelled ACTH analog occurring in assay tubes decreased significantly with gestational age, such differences in degradation could not account for the measured increase in binding at day 60. The maximal number of binding sites measured by the few usable Scatchard analyses obtained were not significantly different between days 60 and 105-undoubtedly reflecting the small sample sizes. However, as with binding to the nonsaturating concentration there was a trend toward a decrease in the number of receptors present. Regulatory factors associated with the reduction in binding of ACTH to its receptors (fmol/pg DNA or pg protein) in the porcine fetus at midgestation must remain speculative. Because ACTH in some species modulates its own receptors (Durand and Locatelli, 1980; Durand et al., 1981; Chatelain et al., 1989; Penhoat et al., 1989), its potential involvement must be considered. There is a preparturient rise in plasma ACTH in pigs (Silver and Fowden, 1989) as has been demonstrated in sheep (Challis and Brooks, 1989). However, in pig fetuses at 70-85 days, plasma ACTH was low (38 f 5 pg/ml) (Silver and Fowden, 1989). Since other published reports for fetal ACTH in pigs deal only with levels at days loo-103 (Brenner et al., 1978; Spencer et al., 1983), additional measurements around day 60 need to be made to substantiate a putative role for ACTH in the increased concentrations of adrenal ACTH receptors. Similarly, the functional significance of the decrease in binding to adrenal ACTH receptors between 60 and 75 days of gestation needs to be determined. Responsiveness of the porcine fetal adrenal has been determined only as early as 89 days of gestational age (Lohse and First, 1981). The entire adrenal gland, not just the cortex, was used for receptor assays. Therefore, it is possible that decreased ACTH binding occurring between 60 and 75 days of gestation reflects an increase in the percent of medullary contribution to the total adrenal mass. Unfortunately, there have been no studies with pigs that have measured the relative contributions of

Fetal porcine adrenal ACTH receptors

the cortex and medulla to the adrenal throughout gestation. In the current studies at 60 days, fetal crown-to-rump length was 138 mm, and at 75 days, 181 mm. It is known that the adrenal cortex anlage appears much earlier than that of the medulla (8 mm stage vs 30-45 mm) (Whitehead, 1903; Weymann, 1922). By the 142 mm stage all medullary cells have migrated to the central part of the adrenal, and by 2 18 mm there is a typical adrenal medulla (Weymann, 1922). One can but speculate that between the 138 and 181 mm stage the ratio of medullary to cortical tissue changes or remains constant. In sheep, this ratio is constant from 90 days to birth at 146 days (Coulter et al., 1989). However, since species differences do exist (Jost, 1966), extrapolation from other species is difficult. Data previously reported concerning fetal adrenal ACTH receptors pertained to sheep (Durand, 1979; Durand et al., 1981), humans (Jaffe, 1982), and rats (Chatelain et al., 1989). Studies with sheep and rats used an iodinated ACTH(l-24) preparation which had a reduced biological activity, and led to the characterization of fetal ACTH receptors having a very low affinity (K, = 4.6 x lo6 M-‘) and a large capacity (6-14 nmol/mg protein). Receptors were measured during the last 20% of pregnancy, and in both species dramatic increases in concentration (nmol/pg DNA) and total content (nmol/paired adrenals) of receptors occurred during the last 3-4 days prior to parturition. In sheep, there were no significant changes in ACTH receptor concentrations between 22 and 9 days prior to parturition (Durand, 1979). Such an interval for sheep corresponds to the 90-105 days of gestational age for pigs in the present study, wherein no changes in binding of ACTH to adrenal ACTH receptors was detected. On the contrary, studies with human fetal adrenals made use of the currently used iodinated ACTH analog, and measured ACTH receptors with a more nearly physiological affinity (K, = IO9M-‘; Jaffe, 1983). This latter study made limited measures of ACTH receptors near midpregnancy. It has long been known that adrenal glands do not store large quantities of steroid, and glandular content closely approximates synthesis (Vogt, 1943), and in many but not all circumstances, secretion (Stockham, 1964). Indeed, in the present study of prenatal pigs, the total adrenal cortisol at days 90-105 paralleled previously reported total in vitro secretion of cortisol in porcine fetuses at similar gestational ages (Lohse and First, 1981). The unaltered concentration of adrenal cortisol from days 60 to 90 suggests that cortisol production per adrenal cell remains constant during that time, whereas the increased total content of adrenal cortisol reflects the increased number of cells. This is in contrast to ovine fetal adrenal cells that have highest basal cortisol production on gestational day 50 and again near term (145 days; Glickman and Challis, 1980). In summary, there are prenatal changes in porcine adrenal ACTH receptors that involve numbers and not affinity of the receptors. Concentrations of these receptors are elevated near midgestation and decrease until 9 days prior to term. Because of the constant DNA/protein ratio at this time and increase in total DNA, it is suggested that at this time there is a

83

decrease in the number of receptors per cell. Adrenal cortisol increases between days 90 and 105 of gestation, when adrenal ACTH receptors are at their lowest concentration. Therefore, if an increase in adrenal responsiveness to fetal ACTH accounts in part for increased adrenal cortisol, this change in responsiveness is unlikely to involve the ACTH receptors. authors wish to express their sincere appreciation to Dr J. Ramachandran for the gift of ACTH analog and for technical advice during the validation of the ACTH receptor assay; to David Sypherd for technical assistance during the conduct of these studies; and to Linda Pamell for secretarial assistance during the preparation of this manuscript. This work was supported in part by USDA Competitive Research Grant No. 86-CRCR-I-1951 to H. G. Klemcke. Acknowledgements-The

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