regulation of steroidogenic enzymes metabolizing testosterone or dihydrotestosterone by human chorionic gonadotropin in Differential

cultured

rat

neonatal interstitial cells

Eisuke P. Murono Research Service, Dorn Veteran's Hospital and Departments of Medicine and Physiology, Medicine Columbia, South Carolina, USA

Abstract The present studies examined the effects of hCG on steroidogenic enzyme activities involved in the metabolism of testosterone or dihydroststosterone in cultured rat neonatal interstitial cells. 5\g=a\-reductaseand 17\g=b\\x=req-\ hydroxysteroid dehydrogenase activities, which are involved in the conversion of testosterone to dihydrotestosterone and androstenedione, respectively, were low in cultured neonatal interstitial cells, were unresponsive to hCG and declined to undetectable levels during 14 days of culture. However, \g=D\5-3\g=b\-hydroxysteroid dehydrogenase-isomerase activity, which is involved in the biosynthesis of testosterone, and 5\g=a\-androstane-3\g=a\-hydroxysteroid dehydrogenase and 5\g=a\-androstane-3\g=b\-hydroxysteroid dehydrogenase activities, which are involved in the conversion of dihydrotestosterone to 5\g=a\-androstan\x=req-\ 3\g=a\,17\g=b\-dioland 5\g=a\-androstan-3\g=b\,17\g=b\-diol,respectively, were maintained or increased by hCG during the same culture period. These results demonstrate that 5\g=a\-reductase and 17\g=b\-hydroxysteroiddehydrogenase activities do not play a significant role in regulating testosterone accumulation in fetal/neonatal Leydig cells, and they suggest that these cells are adapted to maintain high testosterone but low dihydrotestosterone levels. The present results demonstrate also that fetal/neonatal Leydig cells differ from immature or adult Leydig cells with respect to the sensitivity of 5\g=a\-reductaseactivity to LH/hCG.

Fetal Leydig cells in the rat appear around day 15-16 of gestation and begin to secrete testoste¬ rone, which stabilizes the male reproductive tract and accessory sexual organs (1). Some studies sug¬ gest that their appearance and secretion of testoste¬ rone is regulated by LH from the fetal pituitary

University of South Carolina School of

gland (2,3) which is elevated during this period of development (4). However, others suggest that this process is independent of external regulatory con¬ trol, and is genetically programmed to occur (5,6). Nevertheless, fetal or neonatal Leydig cells re¬ spond to exogenous LH/hCG by increased testoste¬ rone

formation

(7,8),

and cultured fetal/neonatal

Leydig cells retain sensitivity to LH/hCG or cAMP

for two or more weeks in culture (9,10). Afterbirth, fetal Leydig cells regress and circulating testoste¬ rone levels decline (11,12). Starting around the second or third week following birth, a second ("adult") generation of Leydig cells appear (13). Leydig cells from immature rats (20-40 days of age) actively synthesize testosterone; however, very little androgen is secreted because high 5ot-reductase ac¬

tivity converts testosterone to dihydrotestosterone (DHT), 5ct-androstan-3a,17ß-diol (3ct-diol) and 5ct-androstan-3ß,17ß-diol (3ß-diol) (14-16). Testo¬ sterone becomes the primary androgen secreted by

adult Leydig cells because of the selective decline in 5a-reductase activity (15). Thus, 5a-reductase ac¬ tivity plays a key role in regulating testosterone ac¬ cumulation and secretion by Leydig cells. Previous studies have demonstrated that the major androgen produced by fetal Leydig cells is testosterone and that very little DHT is secreted (17,18). This implies low 5ct-reductase activity in fetal Leydig cells. 5a-reductase activity is stimu¬ lated by LH/hCG in testes of both immature (19-21) and adult (22) hypophysectomized rats. The

were recrystallized prior to use. thin-layer chromatography sheets (ITLC) were from Gelman Sciences, Ann Arbor, MI. 4-methyl-4-aza3-oxo-pregnan-20(s)-carboxylate (4-MAPC) was a gift from Merck, Sharpe and Dohme, Rahway, NJ.

present studies examined whether 5ct-reductase ac¬

loids, Wilton, NH and

hydroxysteroid dehydrogenase-isomerase (A3-3ßHSD) activity which is increased in cultured neo¬

Isolation

tivity of fetal/neonatal rat testes is sensitive in a sim¬ ilar manner to LH/hCG. The response of 5cx-reductase activity to hCG in cultured interstitial cells from neonatal rats was compared to that of AD-3ßnatal interstitial cells (10). Because testosterone levels can be affected by 17ß-hydroxysteroid dehy¬ drogenase (17ß-HSD) activity, which converts testosterone to A4-androstenedione, the effect of hCG on this enzyme was examined. In addition, because DHT levels can be influenced by 5a-an-

drostane-3a-hydroxysteroid dehydrogenase (5a-

ane-3a-HSD) and 5a-androstane-3ß-hydroxysteroid dehydrogenase (5ct-ane-3ß-HSD) activities, convert DHT to 3a- and 3ß-diol, respec¬ the effects of hCG on these enzymes were examined.

which

tively,

Animals

Pregnant Sprague-Dawley rats were obtained from ZivicMiller Laboratories, Zelienople, PA. They were main¬ tained at 22°C with a controlled light cycle (12 h light and 12 h dark) and provided dry rat chow and tap water ad libitum. Male pups were 4-5 days of age at the time of sacrifice; they were first rendered unconscious with C02, then decapitated between 08.00 and 09.00 h. Testes were quickly removed and maintained at 4°C.

Reagents

Collagenase (Type I), hCG, dimethyl sulfoxide, nitroblue tetrazolium, etiocholanolone, spironolactone, penicillin G streptomycin

Co, St. Louis, MO.

purchased

purchased from Sigma Chemical Reagent grade organic solvents were

were

from Fisher Scientific CO, Atlanta, GA. Dulbecco's modified Eagle's medium (DMEM), Ham's F-12 nutrient mixture (F-12), NaHCOs and HEPES buffer were purchased from Grand Island Biological Company, Grand Island, NY. [7-3H]pregnenolone (23 Ci/mmol),

mCi/mmol), [l,2,6,7,-3H]testo(99 Ci/mmol), [7-3H]testosterone (25 Ci/mmol), [4-14C]androstenedione (52 mCi/mmol), [methyl-3H] thymidine (6.7 Ci/mmol), and protosol were from DuPont

[4-,4C]progesterone (57

sterone

Co, Boston, MA. 5rx-dihydro [la,2ct(n)-3H]testosterone (54 Ci/mmol), 5a-[la,2a(n)3H]androstane-3ß,17ß-diol

(40 Ci/mmol), and 5a-[la,2a(n)-3H]androstane-3a, 17ßdiol (40 Ci/mmol) were from Amersham Corporation, Ar¬ lington Heights, IL. The purity of each labelled steroid was assessed by chromatography or recrystallization. Unlabelled pregnenolone, progesterone, testosterone, androstenedione, dihydrotestosterone, 5a-androstan-3a,

17ß-diol and 5a-androstan-3ß,17ß-diol were

from Stera-

of interstitial cells

experiment testes were obtained from 20 to 40 4-5-day-old male pups. Testes were decapsulated and in¬ cubated for 40 min at 37°C with 0.025% collagenase dis¬ solved in a 1:1 mixture of DMEM/F-12 containing 15 mmol/1 HEPES, 14 mmol/1 NaHC03, 169 nmol/1 penicillin G, and 69 nm°l/l streptomycin. Following digestion dis¬ persed cells were filtered through a nylon mesh to sepa¬ rate undissociated tubules. The dispersed interstitial cells were washed thrice with fresh media and resuspended in DMEM/F-12 containing 15 mmol/1 HEPES, 14 mmol/1 NaHC03, 169 nmol/1 penicillin G, and 69 nmol/1 strep¬ tomycin to give a final concentration of 1.2-1.5-108 cells/1. For each

Based on trypan blue exclusion, it was estimated that over 80% of the dispersed cells were viable.

Culture

Materials and Methods

and

Instant

of dispersed interstitial cells

One ml of cells was plated into each 16-mm diameter Costar culture well (24-well culture plate, Cambridge, MA). Treatment was initiated on the day of plating. Media were changed and cells retreated on day 3 of cul¬ ture. Thereafter, media were changed and cells retreated every other day, for a total of 14 days in culture. Cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% C02. Media were saved on days 3, 7 and 14 of culture for measurement of testosterone by radioimmunoassay as described previously (23). On the same days, intact cells were assayed for various steroido¬ genic enzyme activities.

Assay of As-3$-HSD activity in cultured interstitial cells A -3ß-HSD

activity of cultured interstitial cells was esti¬ mated using a method similar to that described for cul¬ tured murine Leydig cells (24), with the following mod¬ ifications: 1. cells were pre-incubated for 30 min with fresh culture media instead of 1 h, then washed two ad¬ ditional times with fresh media to remove endogenous substrate, and 2. during the actual incubation, 10 nmol/1 spironolactone was added to inhibit 17ct-hydroxylation of the substrate pregnenolone and the product progeste¬ rone (25). In preliminary studies it was demonstrated that activity was linear for up to 1 h and for the number of cells incubated. For the actual assay [ H]pregnenolone (2 nmol/1, 0.5 nCi) and 10 nmol/1 spironolactone were added in culture media containing dimethyl sulfoxide (0.3% final concen¬ tration) to initiate the reaction. The reaction was for 1 h at 37°C in a humidified atmosphere of 95% air and 5% C02. The reaction was terminated by the addition of 0.1 ml of 1 mol/1 NaOH. One hundred ng each of pregne

nolone and [14C]progesterone (~ 4000 cpm) were added carriers and to estimate recoveries. Samples were ex¬ tracted with 5 vol diethyl ether, and the residues of ether extracts were chromatographed by ITLC using chloroform:methanol (99.5:0.5, v/v) as the solvent system. The product, progesterone, was localized using iodine vapors, which was cut out and counted in Omnifluor-toluene. Recovery estimates were approximately 60%.

Results

as

5a-reductase assay 5a-reductase activity was estimated on cultured cells using methods similar to that described for A3-3ß-HSD activity except the reaction was initiated by the addition of [3H]testosterone (10 nmol/1, 0.5 nCi) dissolved in culture media containing 0.3% dimethyl sulfoxide (final concen¬ tration). The reaction was for 1 h at 37°C and was termi¬ nated by the addition of 0.1 ml of 1.0 mol/1 NaOH. Unlabelled testosterone, DHT, 3a- and 3ß-diol (100 ng each) were added to serve as carriers and about 4000 cpm 14C-DHT to estimate recovery. In separate tubes about 4000 cpm of [3H]3a-diol or [3H]3ß-diol were added to estimate recovery for these steroids. Samples were ex¬ tracted with diethyl ether, and the ether-extracted resi¬ dues were processed by ITLC to isolate DHT and 3a- and 3ß-diol. 5a-reductase activity is presented as nmol (DHT + 3a-diol + 3ß-diol) h"1 (105 cells)"1. •

Effect of hCG on testosterone levels On day 3 of culture testosterone level in

media of control neonatal interstitial cells was 13.0 ± 0.3 pmol (10° cells)-1 (Fig. 1). In the presence of 1 or 10 mlU hCG, testosterone levels increased to 38.7 ± 3.1 pmol (p