J O U R N A L OF U L T R A S T R U C T U R E R E S E A R C H
69, 98-108 (1979)
Functional and Ultrastructural Changes during ACTH-Induced Early Differentiation of Cortical Cells of Human Fetal Adrenals in Primary Cultures R . VOUTILAINEN AND A. I. KAHRI 1
Department of Pathology, University of Helsinki, Helsinki, Finland Received March 29, 1979 Early events during ACTH-induced differentiation of cortical cells of human fetal adrenals in primary cultures were studied by electron microscopy and steroid analysis. In undifferentiated cortical cells, the basal secretion of steroids was very low. The first sign of ACTH stimulation was a marked increase in pregnenolone sulfate secretion during the first 24 hr, indicating the activation of "pregnenolone synthetase" enzyme. The main shift towards differentiated function occurred during the second and third days of stimulation, shown by increased secretion of 11-deoxycortisol, corticosterone, cortisol, and dehydroepiandrosterone sulfate, while the pregnenolone sulfate concentration decreased. Aldosterone secretion increased only very slightly and slowly during ACTH stimulation. The first change in the morphology of cortical cells was the rounding of the cells during the first 24 hr. Ultrastructurally this was seen by a transformation of plasma membrane into microvillous projections and a loosening of the cell connections. The main functional activation of cortical cells was reflected later as increased smooth endoplasmic reticulum membranes.
The proliferative cell type in the human fetal adrenal cortex in tissue culture has been found to be the zona glomerulosa or permanent cortex type cortical cell (Kahri and Halinen, 1974). These morphologically undifferentiated cells have been found to have a very small capacity of 17a- and llflhydroxylations (Kahri et al., 1976; Voutilainen, 1979). In the presence of ACTH these undifferentiated cortical cells are able to differentiate into zona fasciculata like cells, which have a high capacity of secreting cortisol and corticosterone (Kahri et al., 1976; Voutilainen, 1979). The ultrastructural characteristics of these differentiated cells are the same as those of adult zona fasciculata cells: a large amount of smoothsurfaced endoplasmic reticulum membranes, some groups of lamellar rough endoplasmic membranes, dense groups of polysomes, an increase in the storage of lipids, and development of microvilli. The ultrastructural features induced by ACTH in tissue culture of human fetal adrenals ~Supported by a grant from the Sigrid Jus6hus Foundation, Helsinki, Finland. 98 0022-5320/79/100098-10502.00/0 Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
have to be comparable with the in vivo situation (Kahri et al., 1976). Due to the absolute impossibility of obtaining experimental information on the early events in the differentiation of cortical cells of human adrenal cortex in vivo, primary tissue culture of human fetal adrenals has been used in the present study to clarify these events during ACTH-induced differentiation of the zona glomerulosa-like cells into zona fasciculata-like cells. The main interest has been directed to the first, second, and third days of ACTH exposure, which have been found to be the most critical days in the differentiation of the cortical cells of rat adrenals in tissue culture (Kahri et al., 1970; Salmenper~i and Kahri, 1977). The ultrastructure and the steroidforming capacity of cultivated cortical cells of human fetal adrenals was studied both in the unstimulated state and at different times after ACTH stimulation. The steroids secreted into the tissue culture medium were analyzed using gas-liquid chromatography for cortisol, corticosterone, ll-deoxycortisol, dehydroepiandrosterone sulfate,
DIFFERENTIATION OF HUMAN FETAL ADRENALS
and pregnenolone sulfate, and radioimmunoassay for aldosterone. MATERIALS AND METHODS
Tissue Culture and Electron Microscopy The tissue culture method previously found to be suitable for long-term cultivation of fetal rat adrenals was used in this study (Kahri, 1966). Human early midterm fetuses were obtained from abortions induced for sociomedical reasons. The fetuses were delivered by abdominal hysterotomy. The adrenals were removed aseptically and transported to the laboratory in ice-cold Hanks' BSS (balanced salt solution). The crown-rump lengths of the fetuses used in this study were 9.5-17 cm, corresponding to the gestational age of 13-18 weeks (Tanimura et al., 1971). The adrenal tissue amount per culture dish was about 15 mg. The tissue culture medium consisted of 50% Melnick's solution A (0.5% lactalbumin hydrolysate in Hanks' BSS), 25% Eagle's minimum essential medium (both from Pharmaceutical Manufacture Orion Oy, Finland), and 25% heat-inactivated and ultraffltered newborn calf serum (Gibco, United Kingdom). Five milliliters of medium were used per culture. During cultivation, morphology and cell viability were monitored by phase contrast microscopy. The ACTH used was Synacthen (Ciba-Geigy Limited, Basel, Switzerland) and it was added at a rate of 0.2/~g/ml (= 20 mIU/ml) of tissue culture medium per day. This dose causes maximal stimulation of steroidogenesis in the present system, Dibutyryl cyclic AMP (dbcAMP) was from Sigma Chemicals (St. Louis, Mo.) and it was added at a rate of 100 ttg/ml of tissue culture medium per day. After experiments the culture media were removed and stored at -20°C until analyzed. The cultures were then fixed in situ in their dishes with 2.5% glutaraldehyde (Electron Microscopy Sciences, Fort Washington, Pa.) in Hanks' BSS (the pH of the fixative was adjusted to 7.3 at the beginning of the fixation). Postfixation was done in osmium tetroxide in a phosphate buffer (Millonig, 1962). Dehydration was accomplished using a graded ethanol series after which the cultures (still in situ) were embedded in a mixture of Araldite (Ciba, USA) and Epon 812 (Shell, USA). The sections were stained with 0.2% lead citrate (Venable and Coggeshall, 1965) and examined with a Hitachi HS-7S electron microscope.
Steroid Analysis The procedure for the extraction and purification of the steroids was essentially the same as described earlier (Salmenper/i and Kahri, 1976) with slight modifications. The method is described in detail elsewhere (Voutilainen, 1979). Briefly: After addition of radio~ active steroids as recovery tracers, the media were extracted with 10 vol of dichloromethane. The extracts
were washed with 1/10 vol of 0.1 N NaOH and twice with this volume of distilled water. After drying under nitrogen the samples were partitioned between 80% ethanol and cyclohexane 1/4 to reduce the lipid load. Purification on 0.5-g Sephadex LH-20 columns was then performed. The water residue of the primary dichloromethane extraction was used for analyzing sulfate conjugates. Solvolysis was performed as described earlier (Burstein and Lieberman, 1958; Vihko, 1966). Hi-Flosil column chromatogr,aphy was used for purification of the solvolysed sulfate conjugates. The steroids (except aldosterone) were analyzed by gasliquid chromatography on 1% SE-30 columns as their O-methyloxime-trimethylsilyl ethers. A flame ionization detector was used and the responses were recorded with a digital integrator. Aldosterone was measured by radioimmunoassay using a highly specific antiserum (New England Nuclear, Mass.).
Development of the Steroidogenic Capacity of Human Fetal Adrenals in Primary Cultures during ACTH-Induced Differentiation Chart 1 shows the development of the steroidogenic capacity of cultivated fetal adrenal cortical cells in respect to the duration of ACTH stimulation. The first values of the chart show the predominance of sulfate conjugates during the first cultivation day. This represents secretion of the fetal zone cells which later disappear from the cultures (Kahri et al., 1976). After stimulating the culture for 24 hr, the only change in the steroid pattern was a remarkable increase in pregnenolone sulfate production, while DHAS (dehydroepiandrosterone sulfate), aldosterone and cortisol did not increase significantly at this stage. As ACTH stimulation was continued, the cortisol and DHAS secretion capacity gradually increased from the second day of stimulation onwards at the same time as pregnenolone sulfate secretion decreased. The increase in aldosterone secretion was clearly less than in cortisol and DHAS secretion during ACTH stimulation. Table 1 shows cumulative secretion of cortisol, 11-deoxycortisol, and corticosterone after various periods of ACTH stimu-
VOUTILAINEN AND KAHRI
DHAS 3 PregnS
F Aldos, DHAS
#g/5 mb 2
Aldost ng/Sml 0.3 0.2 01
7 8 9 10 DAYS OF CULTIVATJON
ACTH 01 lU/5ml/day
CHART 1. The development of steroidogenic capacity of cultured human fetal adrenals during ACTH stimulation. The steroid production of the same culture was analyzed at various stages of cultivation. The medium was changed daily. F, cortisol; Aldost, aldosterone; DHAS, dehydroepiandrosterone sulfate; PregnS, pregnenolone sulfate. lation. T h e table shows t h a t the relative secretion of 11-deoxycortisol in respect to cortisol is greater during the first three stimulation days t h a n later (see changes in c o r t i s o l / l l - d e o x y c o r t i s o l ratio). T a b l e 2 shows the effects of A C T H and d b c A M P on c u m u l a t i v e steroid production during the 6th to 12th days of cultivation (the cultures are not f r o m the s a m e fetus as those of T a b l e i and C h a r t 1); d b c A M P h a d qualitatively the s a m e effect on steroidogenesis as A C T H .
Ultrastructure of Cortical Cells of Human Fetal Adrenals in Primary Cultures during ACTH-Induced Early Differentiation
s m o o t h - s u r f a c e d endoplasmic reticulum m e m b r a n e s were still absent and only few t u b u l a r rough endoplasmic reticulum m e m branes were visible in the c y t o p l a s m (Fig. 3). T h e m i t o c h o n d r i a h a d lamellar inner m e m b r a n e s and were in the s h a p e of elongated ellipsoids with n u m e r o u s dense matrix granules. S o m e lipid droplets were always visible in the cytoplasm. The 2nd day. At this stage of stimulation the cytoplasmic organelles were still poorly developed (Fig. 4). R o u g h endoplasmic reticulum m e m b r a n e s and rich ribosomal granules and p o l y s o m e s b e c a m e increasingly n u m e r o u s and m o r e prominent. S m o o t h - s u r f a c e d endoplasmic reticulum m e m b r a n e s were still absent. S o m e mitochondria h a d a greatly increased matrix v o l u m e with a few dense granules in the matrix a n d the inner m e m b r a n e s were lamellar. T h e r e was no increase in the number of lipid droplets. The 3rd day. T h e m o s t p r o m i n e n t feature at this stage of differentiation was the increase in cytoplasmic volume and the large a m o u n t of s m o o t h endoplasmic reticu l u m m e m b r a n e s in the c y t o p l a s m (Fig. 5). Dense groups of lamellar rough m e m b r a n e s and groups of polysomal aggregates were also a typical feature of cortical cells stimulated for 3 days with A C T H . T h e r e was an increase in mitochondrial profiles in the
The 1st day. After stimulating the cortical cells for 24 hr with A C T H the m o s t drastic event was the change in s h a p e f r o m flattened cuboidal cells into m o r e spherical ones, and the s e p a r a t i o n of the cortical cells f r o m each other. Ultrastructurally, this was found to follow the d e v e l o p m e n t of numerous microvilli of the p l a s m a m e m b r a n e and a decrease in the direct contacts between cortical cells (Fig. 1). T h e o t h e r ultrastructural differences at this stage of developm e n t r e m a i n e d scarce w h e n c o m p a r e d with the cortical cells of the p e r m a n e n t cortex at the beginning of stimulation (Fig. 2). T h e
TABLE 1 CUMULATIVE CORTISOL, ll-DEoxYCORTISOL AND CORTICOSTERONE SECRETION AT VARIOUS TIMES AFTER A C T H STIMULATIONa
Cortisol 11-Deoxycortisol Corticosterone Ratio cortisol/lldeoxycortisol
Control 1 day 20