IN TISSUE CULTURE OF FETAL STEROIDOGENESIS AND ADULT HUMAN ADRENALS WITH SPECIAL REFERENCE TO CORTISOL/CORTICOSTERONE RATIO AND ALDOSTERONE SECRETION Department
RAIMO VOUTILAINEN of Pathology, University of Helsinki, SF-00290 Helsinki 29, Finland (Received 16 June 1978) SUMMARY
Simultaneous analysis of the main free and sulphate-conjugated adrenal steroids (cortisol, corticosterone, lldeoxycortisol, aldosterone, dehydroepiandrosterone. androstenedione, 18-hydroxydeoxy corticosterone (II-OH-DOC), dehydroepiandrosterone sulphate and pregnenolone sulphate) secreted by human fetal and adult adrenals in tissue culture was performed. Sephadex LH-20 column chromatography was used for fractionation of the free steroids. The “androgen fraction” from Sephadex column and the solvolysed sulphate conjugates were further purified by Hi-Flosil column chromatography. Aldosterone was quantitated using the radioimmunoassay, the other steroids were measured by g.1.c. During the first days of cultivation fetal adrenals secreted mainly sulphate conjugates of 3/?-hydroxy-S-ene steroids and during later cultivation steroidogenesis clearly decreased. ACTH treatment at this later stage markedly stimulated steroidogenesis and altered the steroid pattern towards the adult type. No specific block in aldosterone biosynthesis was noted in fetal cultures. l&OH-DOC was not discovered in either fetal or adult cultures by g.1.c. The F/B ratio (cortisol/corticosterone) in adult cultures was constantly clearly smaller than in fetal cultures. The F/S ratio (cortisol/l ldeoxycortisol) decreased when corticosterone concentration increased in adult cultures. This is probably due to the inhibitory effect of corticosterone on llg-hydroxylation.
INTRODUCTION
The steroidogenic capacity of human adrenals during fetal [e.g. l-51 and adult life [e.g. 61 is described in the literature. The main difference between adult and fetal adrenal cortex is the lack of enzymatic activity of the 3B-hydroxy-steroid dehydrogenase 5-ene + 4ene isomerase system in fetal adrenals. The dependence of the differentiation and the steroid secretion capacity of cortical cells of human fetal adrenals on ACTH has been described [7]. However, there are still unsolved problems concerning the relative distribution of certain steroids secreted (e.g. l&OH-DOC, aldosterone, 1l-deoxycortisol and corticosterone in respect to cortisol) and their physiological role in general and in the maturation of adrenal cortex. The purpose of the present study was to measure simultaneously the main free and sulphate-conjugated steroids in tissue culture of human adrenals and to map the differences of fetal and adult steroidogenesis. The main specific aims were to determine aldosterone and 18-OH-DOC secretion in fetal adrenals and to compare the F/B (cortisol/corticosterone) and F/S (cortisol/l Ideoxycortisol) secretion ratios in adult and fetal adrenal cortex in primary culture. EXPERIMENTAL Chemicals
All solvents were of reagent grade and were redistilled before use. Dichloromethane, reagent grade, was S.B. IO/l-l
115
washed 3 times with distilled water, stored overnight over sodium hydroxide flakes, distilled and stored in aliquots at - 20°C. Silylating reagents hexamethyldisilazane and trimethylchlorosilane were obtained from Fluka AG (Switzerland). Methoxyamine hydrochloride was obtained from Eastman Organic Chemicals (Rochester, NY). Sephadex LH-20 was purchased from Pharmacia (Uppsala, Sweden). Hi-Flosil column chromatography support was obtained from Applied Science Laboratories (State College, PA). Albumin (fraction 5) was obtained from Armour (Eastbourne, England). Norit A neutral charcoal and Dextran T-70 were purchased from Amend (Irvington, England) and from Pharmacia, respectively. Antiserum for aldosterone assay was obtained from New England Nuclear (MA). ACTH used was Synacthen (Ciba-Geigy Ltd, Base], Switzerland). Dibutyryl cyclic AMP (dbcAMP) was from Sigma Chemical Co. (St. Louis, MO). Reference steroids Radioactive steroids. [1,2-3H]-Corticosterone, [1,2-3H]-18-hydroxy-deoxycorticosterone (18-OHDOC, 18,21dihydroxy-4-pregnene-3,20-dione) and [1,2-3H]-aldosterone were obtained from the Radiochemical Centre (Amersham, England). [1,2-3H]-Cortisol, [7-3H]dehydroepiandrosterone (DHA, 3&hydroxy-5-androsten-17-one) and [7-3H]dehydroepiandrosterone sulphate were purchased from NEN Chemicals GmbH (Dreieichenhain, West Germany).
RAIMO VOUTILAINEN
116
Non-radioactive steroids. These were purchased mostly from Sigma Chemical Co. Other sources were: Steraloids (NY): progesterone; Koch-Light (Colnbrook, England): cholesteryl butyrate (3fl-hydroxycholest-S-ene-3-n-butyrate) and 1 lfl-OH-androstenedione (lll(-hydroxy-4-androstene-3,17-dione); E. Merck (Darmstadt. West Germany): pregnenolone. Tissue
and tissue
culture
Human early mid-term fetuses were obtained from abortions induced for sociomedical reasons. The fetuses were delivered by abdominal hysterotomy and immediately bled from the umbilical cord. The adrenals were removed aseptically and transported to the laboratory in ice-cold Hanks’ balanced salt solution. The crown-rump length of the fetus used in this study was 13 cm.. corresponding to the gestational age of 15 weeks [S]. Adult adrenals were obtained from surgery. One of the adrenals used in this study was from a patient having Cushing’s syndrome owing to mild bilateral hyperplasia of adrenals, the other adrenal used was normal. The method of culturing fetal rat adrenals described by Kahri[9] was used. The medium (5 ml/culture dish), consisting of 500/b Melnick’s solution A (O.So/, lactalbumin hydrolysate in Hanks’ balanced salt solution), 25”~; Eagle’s Minimum Essential Medium (both from Pharmaceutical Manufacture Orion Oy, Finland) and 259/, newborn calf serum (heat inactivated) (Gibco, England) was replaced every 6th day. The tissue amount per culture dish was about 1&20mg and in each culture group we tried to explant equal amounts of adrenal tissue on parallel dishes. Because the main purpose of the study was to get a view of relative secretion of certain steroid, it was not essential to know the exact amou. of tissue per dish. Steroid
extraction
and pu@cation
of [l.2-3H]-aldosterone, About 1500 c.p.m. [1,2-3H]-corticosterone, [1.2--‘HI-cortisol, [7-jH]dehydroepiandrosterone in 50~1 of ethanol and [7-3H]-dehydroepiandrosterone sulphate in 50 nl of methanol were added to the tissue culture medium as recovery tracers. The used procedure for extraction and purification of free steroids was essentially the same as describea earlier [lo] with slight modifications. Briew ,he media were extracted with 10 vol of dichloromethane. The extracts were washed with l/l0 vol 0.1 N NaOH and twice with this volume of distilled water. After drying under nitrogen the samples were partitioned between SO”: ethanol and cyclohexane (1 :4) to reduce the lipid load. The ethanol phase was extracted with 2 vol of dichloromethane and the remaining water phase with 10 vol of dichloromethane. The combined extracts were dried under nitrogen and the Sephadex LH-20 column chromatography was performed. For conditions and collected fractions see Fig. 1. The water residue of the primary dichloromethane extraction and the NaOH washing water were combined and
used for analysing sulphate conjugates. Solvolysis was performed as described earlier [ll. 123. Hi-Flosil column chromatography was used for purification of the solvolysed sulphate conjugates (for conditions see Fig. 1). Similar Hi-Flosil columns were used for further purification of fraction I (“the androgen fraction”) from the Sephadex column, because the fraction I was too impure to allow reliable quantitation of androgens on the gas chromatogram. Gas-liquid
chromatography
(g.l.c.)
Cholesteryl butyrate (1.25-10.0 pg) was added in 100 ~1 of toluene to ethanol dissolved residues of fractions II and IV from the Sephadex column after taking one fourth for recovery analysis. Estriol (1.25-5.0 pg) was added respectively as an internal standard to ethanol-solved residues of fractions collected from the Hi-Flosil column (“androgen” and “sulphate” fractions). MO-TMS (O-methyloxime-trimethyl-silyl ether) derivatives were prepared essentially as described earlier [lo, 131. A Perkin-Elmer F-30 gas chromatograph with 1% SE-30 packed columns was used. The flame ionization responses were recorded with a digital integrator, and concentrations were calculated based on the responses of the peaks of the analysed steroids respective to the response of the standard. The results were corrected for methodological losses with recovery indicators. RESULTS
Methodolog!
The percentual recoveries of the added radioactive tracers were: aldosterone 55.4 k 6.4, dehydroepiandrosterone 68.6 +_ 3.4. cortisol 89.1 f 4.6, corticosterone 81.7 + 5.6, and dehydroepiandrosterone sulphate 73.6 + 8.4 (means + SD.). Only one recovery tracer was used per fraction collected from the columns. The recovery of other steroids was tested with non-radioactive standards added to pure tissue culture medium. The fractional recovery of 1 l-deoxycortisol was found to be constantly of the same magnitude as that of corticosterone (about 80’;), so the percentual recoveries of corticosterone were used to correct the methodological losses of 11-deoxycortisol. In the same way the values of pregnenolone sulphate were corrected according to the recoveries of dehydroepiandrosterone sulphate supposing that solvolysis was as effective for pregnenolone sulphate as for DHA sulphate. The recovery percentage of androstenedione in this procedure was noted to be constantly 7% smaller than that of dehydroepiandrosterone (on an average 62?/,). This was taken into consideration when correcting the values of androstenedione. Blanks from the tissue culture medium (analysed from 20 ml) were clearly smaller than any values represented in Tables 1 and 2. The coefficient of variation of the method for different steroids varied from 2.5 to 17% in the concentration range found in this study.
Steroidogenesis
in cultured adrenals
117
CPM
Hi - Flosil
ELUTION SEPHADEX
VOLUME LH-20
0.15g
ml
I I Methanol
0.59
Fig. 1. The elution diagram of corticosteroids from a OSg Sephadex LH-20 column. Column (I.D. 0.6 cm.) steeled by gravity, swelled in So”/, methanol. Eluant: toluene-cyclohexane (2 : 1) equilibrated overnight with 80”
RAIMO VOUTILAINEN of Pathology, University of Helsinki, SF-00290 Helsinki 29, Finland (Received 16 June 1978) SUMMARY
Simultaneous analysis of the main free and sulphate-conjugated adrenal steroids (cortisol, corticosterone, lldeoxycortisol, aldosterone, dehydroepiandrosterone. androstenedione, 18-hydroxydeoxy corticosterone (II-OH-DOC), dehydroepiandrosterone sulphate and pregnenolone sulphate) secreted by human fetal and adult adrenals in tissue culture was performed. Sephadex LH-20 column chromatography was used for fractionation of the free steroids. The “androgen fraction” from Sephadex column and the solvolysed sulphate conjugates were further purified by Hi-Flosil column chromatography. Aldosterone was quantitated using the radioimmunoassay, the other steroids were measured by g.1.c. During the first days of cultivation fetal adrenals secreted mainly sulphate conjugates of 3/?-hydroxy-S-ene steroids and during later cultivation steroidogenesis clearly decreased. ACTH treatment at this later stage markedly stimulated steroidogenesis and altered the steroid pattern towards the adult type. No specific block in aldosterone biosynthesis was noted in fetal cultures. l&OH-DOC was not discovered in either fetal or adult cultures by g.1.c. The F/B ratio (cortisol/corticosterone) in adult cultures was constantly clearly smaller than in fetal cultures. The F/S ratio (cortisol/l ldeoxycortisol) decreased when corticosterone concentration increased in adult cultures. This is probably due to the inhibitory effect of corticosterone on llg-hydroxylation.
INTRODUCTION
The steroidogenic capacity of human adrenals during fetal [e.g. l-51 and adult life [e.g. 61 is described in the literature. The main difference between adult and fetal adrenal cortex is the lack of enzymatic activity of the 3B-hydroxy-steroid dehydrogenase 5-ene + 4ene isomerase system in fetal adrenals. The dependence of the differentiation and the steroid secretion capacity of cortical cells of human fetal adrenals on ACTH has been described [7]. However, there are still unsolved problems concerning the relative distribution of certain steroids secreted (e.g. l&OH-DOC, aldosterone, 1l-deoxycortisol and corticosterone in respect to cortisol) and their physiological role in general and in the maturation of adrenal cortex. The purpose of the present study was to measure simultaneously the main free and sulphate-conjugated steroids in tissue culture of human adrenals and to map the differences of fetal and adult steroidogenesis. The main specific aims were to determine aldosterone and 18-OH-DOC secretion in fetal adrenals and to compare the F/B (cortisol/corticosterone) and F/S (cortisol/l Ideoxycortisol) secretion ratios in adult and fetal adrenal cortex in primary culture. EXPERIMENTAL Chemicals
All solvents were of reagent grade and were redistilled before use. Dichloromethane, reagent grade, was S.B. IO/l-l
115
washed 3 times with distilled water, stored overnight over sodium hydroxide flakes, distilled and stored in aliquots at - 20°C. Silylating reagents hexamethyldisilazane and trimethylchlorosilane were obtained from Fluka AG (Switzerland). Methoxyamine hydrochloride was obtained from Eastman Organic Chemicals (Rochester, NY). Sephadex LH-20 was purchased from Pharmacia (Uppsala, Sweden). Hi-Flosil column chromatography support was obtained from Applied Science Laboratories (State College, PA). Albumin (fraction 5) was obtained from Armour (Eastbourne, England). Norit A neutral charcoal and Dextran T-70 were purchased from Amend (Irvington, England) and from Pharmacia, respectively. Antiserum for aldosterone assay was obtained from New England Nuclear (MA). ACTH used was Synacthen (Ciba-Geigy Ltd, Base], Switzerland). Dibutyryl cyclic AMP (dbcAMP) was from Sigma Chemical Co. (St. Louis, MO). Reference steroids Radioactive steroids. [1,2-3H]-Corticosterone, [1,2-3H]-18-hydroxy-deoxycorticosterone (18-OHDOC, 18,21dihydroxy-4-pregnene-3,20-dione) and [1,2-3H]-aldosterone were obtained from the Radiochemical Centre (Amersham, England). [1,2-3H]-Cortisol, [7-3H]dehydroepiandrosterone (DHA, 3&hydroxy-5-androsten-17-one) and [7-3H]dehydroepiandrosterone sulphate were purchased from NEN Chemicals GmbH (Dreieichenhain, West Germany).
RAIMO VOUTILAINEN
116
Non-radioactive steroids. These were purchased mostly from Sigma Chemical Co. Other sources were: Steraloids (NY): progesterone; Koch-Light (Colnbrook, England): cholesteryl butyrate (3fl-hydroxycholest-S-ene-3-n-butyrate) and 1 lfl-OH-androstenedione (lll(-hydroxy-4-androstene-3,17-dione); E. Merck (Darmstadt. West Germany): pregnenolone. Tissue
and tissue
culture
Human early mid-term fetuses were obtained from abortions induced for sociomedical reasons. The fetuses were delivered by abdominal hysterotomy and immediately bled from the umbilical cord. The adrenals were removed aseptically and transported to the laboratory in ice-cold Hanks’ balanced salt solution. The crown-rump length of the fetus used in this study was 13 cm.. corresponding to the gestational age of 15 weeks [S]. Adult adrenals were obtained from surgery. One of the adrenals used in this study was from a patient having Cushing’s syndrome owing to mild bilateral hyperplasia of adrenals, the other adrenal used was normal. The method of culturing fetal rat adrenals described by Kahri[9] was used. The medium (5 ml/culture dish), consisting of 500/b Melnick’s solution A (O.So/, lactalbumin hydrolysate in Hanks’ balanced salt solution), 25”~; Eagle’s Minimum Essential Medium (both from Pharmaceutical Manufacture Orion Oy, Finland) and 259/, newborn calf serum (heat inactivated) (Gibco, England) was replaced every 6th day. The tissue amount per culture dish was about 1&20mg and in each culture group we tried to explant equal amounts of adrenal tissue on parallel dishes. Because the main purpose of the study was to get a view of relative secretion of certain steroid, it was not essential to know the exact amou. of tissue per dish. Steroid
extraction
and pu@cation
of [l.2-3H]-aldosterone, About 1500 c.p.m. [1,2-3H]-corticosterone, [1.2--‘HI-cortisol, [7-jH]dehydroepiandrosterone in 50~1 of ethanol and [7-3H]-dehydroepiandrosterone sulphate in 50 nl of methanol were added to the tissue culture medium as recovery tracers. The used procedure for extraction and purification of free steroids was essentially the same as describea earlier [lo] with slight modifications. Briew ,he media were extracted with 10 vol of dichloromethane. The extracts were washed with l/l0 vol 0.1 N NaOH and twice with this volume of distilled water. After drying under nitrogen the samples were partitioned between SO”: ethanol and cyclohexane (1 :4) to reduce the lipid load. The ethanol phase was extracted with 2 vol of dichloromethane and the remaining water phase with 10 vol of dichloromethane. The combined extracts were dried under nitrogen and the Sephadex LH-20 column chromatography was performed. For conditions and collected fractions see Fig. 1. The water residue of the primary dichloromethane extraction and the NaOH washing water were combined and
used for analysing sulphate conjugates. Solvolysis was performed as described earlier [ll. 123. Hi-Flosil column chromatography was used for purification of the solvolysed sulphate conjugates (for conditions see Fig. 1). Similar Hi-Flosil columns were used for further purification of fraction I (“the androgen fraction”) from the Sephadex column, because the fraction I was too impure to allow reliable quantitation of androgens on the gas chromatogram. Gas-liquid
chromatography
(g.l.c.)
Cholesteryl butyrate (1.25-10.0 pg) was added in 100 ~1 of toluene to ethanol dissolved residues of fractions II and IV from the Sephadex column after taking one fourth for recovery analysis. Estriol (1.25-5.0 pg) was added respectively as an internal standard to ethanol-solved residues of fractions collected from the Hi-Flosil column (“androgen” and “sulphate” fractions). MO-TMS (O-methyloxime-trimethyl-silyl ether) derivatives were prepared essentially as described earlier [lo, 131. A Perkin-Elmer F-30 gas chromatograph with 1% SE-30 packed columns was used. The flame ionization responses were recorded with a digital integrator, and concentrations were calculated based on the responses of the peaks of the analysed steroids respective to the response of the standard. The results were corrected for methodological losses with recovery indicators. RESULTS
Methodolog!
The percentual recoveries of the added radioactive tracers were: aldosterone 55.4 k 6.4, dehydroepiandrosterone 68.6 +_ 3.4. cortisol 89.1 f 4.6, corticosterone 81.7 + 5.6, and dehydroepiandrosterone sulphate 73.6 + 8.4 (means + SD.). Only one recovery tracer was used per fraction collected from the columns. The recovery of other steroids was tested with non-radioactive standards added to pure tissue culture medium. The fractional recovery of 1 l-deoxycortisol was found to be constantly of the same magnitude as that of corticosterone (about 80’;), so the percentual recoveries of corticosterone were used to correct the methodological losses of 11-deoxycortisol. In the same way the values of pregnenolone sulphate were corrected according to the recoveries of dehydroepiandrosterone sulphate supposing that solvolysis was as effective for pregnenolone sulphate as for DHA sulphate. The recovery percentage of androstenedione in this procedure was noted to be constantly 7% smaller than that of dehydroepiandrosterone (on an average 62?/,). This was taken into consideration when correcting the values of androstenedione. Blanks from the tissue culture medium (analysed from 20 ml) were clearly smaller than any values represented in Tables 1 and 2. The coefficient of variation of the method for different steroids varied from 2.5 to 17% in the concentration range found in this study.
Steroidogenesis
in cultured adrenals
117
CPM
Hi - Flosil
ELUTION SEPHADEX
VOLUME LH-20
0.15g
ml
I I Methanol
0.59
Fig. 1. The elution diagram of corticosteroids from a OSg Sephadex LH-20 column. Column (I.D. 0.6 cm.) steeled by gravity, swelled in So”/, methanol. Eluant: toluene-cyclohexane (2 : 1) equilibrated overnight with 80”
