Age-related changes in conversion of 5\g=a\-androstan-17\g=b\-ol\x=req-\ 3-one to 5\g=a\-androstane-3\g=a\,17\g=b\-dioland 5\g=a\-androstane\x=req-\ 3\g=b\,17\g=b\-diolby rat testicular cells in vitro R. C. Cochran, A. W. Schuetz and L. L. Ewing Division ofReproductive Biology, Department ofPopulation Dynamics, The Johns Hopkins University, School ofHygiene and Public Health, Baltimore, Maryland 21205, U.SA. Summary. Conversion of labelled 5\g=a\-androstan-17\g=b\-ol-3-one(DHT) by isolated testicular cells from rats of different ages was examined under saturating substrate conditions in vitro (5\p=n-\10\g=m\gDHT/ml in a 24 h incubation). Two detectable metabolites of DHT were produced by testicular cells in vitro, 5\g=a\-androstane\x=req-\ 3\g=a\-17\g=b\-diol(3\g=a\-diol)and 5\g=a\-androstane-3\g=b\,17\g=b\-diol(3\g=b\-diol).Production of these diols during a 24 h period was linear, and the amounts formed were directly related to the cell number. The amount of 3\g=a\-and 3\g=b\-diolsformed by testicular cells of rats of different ages increased from Day 10 to Day 25, then declined. Testicular cells from rats 10 to 20 days of age converted DHT mainly to 3\g=a\-diol,but thereafter 3\g=b\\x=req-\ diol was the predominant testicular metabolite of DHT. Introduction Testosterone is the major metabolite of progesterone in testicular tissue sUce preparations taken from rats aged 0-17 days (Ficher & Steinberger, 1968). Between 17 and 40 days of age the predominant products of progesterone metabolism are 5 -reduced androgens, while only a minimal amount of testosterone can be recovered (Ficher & Steinberger, 1971). After 40 days of age, testosterone again becomes the main androgen formed (Steinberger & Ficher, 1969). This biphasic curve in testosterone synthesis in vitro, with an interim period of relatively high levels of 5a-reduced metabolites, correlates weU with the concentrations of these steroids in testes of rats of different ages (Podestà & Rivaróla, 1974). However, the nature of the age-related changes in the conversion of the immediate 5a- reduced metabolite of testosterone, 5a-androstan-17ß-ol-3-one (DHT) remains equivocal because in previous studies: (1) DHT was not used as the radioisotopic precursor and the availa¬ bility of DHT was dependent upon its formation by 5a-reductase activity (Ficher & Steinberger, 1971; Matsumoto & Yamada, 1973; Rivarola, Podestà & Chemes, 1972); (2) metabolism of the low concentrations of the exogenously added radioisotopic steroid may have been affected by the presence of endogenous steroids (Vinson & Whitehouse, 1969); and (3) with one exception (Matsumoto & Yamada, 1973), 5a-androstane-3a,17ß-diol (3a-diol) has not been distinguished from 5a-andostane-3ß,17ß-diol (3ß-diol) (Ficher & Steinberger, 1971; Coffey, French & Nayfeh, 1971; Rivarola et al, 1972; Podestà & Rivarola, 1974) although the two epimers have very different biological properties (Lubicz-Nawrocki, 1973; WiUiams-Ashman, 1975) and are probably produced by two separate enzymes (Inano, Hori & Tamaoki, 1966; Wilson, 1975). As the cellular site of synthesis of the and ß epimers is unknown, their formation from DHT was studied utilizing a dissociated ceU preparation of testicular tissue from rats of different ages. Downloaded from Bioscientifica.com at 09/28/2021 11:12:05AM via free access Materials and Methods Male Sprague-Dawley rats of various ages were obtained from Flow Laboratories (Rockville, U.S.A.). All animals were kept under conditions of controlled Ught (14 h light/24 h) and temperature (20 ± 1°C). Water and Purina Rat Chow were provided ab libitum. UnlabeUed steroids were obtained from Steraloids and crystaUized to constant melting point before use. [1,2-3H]DHT (sp.act. 40 Ci/mmol) was purchased from New England Nuclear Corporation (Boston, U.S.A.) and purified by thin-layer chromatography before use. Organic solvents utilized for U.c. and extraction of steroids were Nanograde from Malinckrodt (St Louis, U.S.A.). Acetonitrile was obtained from Burdick and Jackson (Muskegon, U.S.A.). Ethyl acetate for the extraction of steroids contained 10~6 M-androstenedione, testosterone, DHT, 3a-diol, and 3 ß-diol. Liquid scintillation cocktail was comprised of toluene, 10% methanol, 0-4% PPO, arid 0-004% POPOP. Counting efficiency with this cocktail in a Beckman LS-250 Uquid scintillation counter was approximately 45%. The counting error in the low range was 7%. Control incubations with [3H]DHT and medium alone and reagent blanks with no added [3H]DHT were always performed. Tissue culture solutions Dulbecco's phosphate-buffered saline (Medium 1), and phosphate-buffered saline lacking Ca2+ and Mg2+ (Medium 2) were prepared as described by Davis & Schuetz (1975). Trypsin (Difco 1:250, Detroit, U.S.A.) was dissolved in Medium 2 to make a 0-1% (w/v) solution. Stock fetal calf serum (Gibco, New York, U.S.A.) was diluted with Medium 2 to obtain a working solution (3% FCS). Complete culture medium (Medium 3) consisted of NCTC 135 (Gibco), 10% fetal calf serum, and 1% penicUlin-streptomycin solution (Gibco). All solutions were sterilized by miUipore filtration. Experiments Animals were weighed, then killed by cervical dislocation. Testes were decapsulated in Medium 2 and dissociated with 0-1% trypsin (Davis & Schuetz, 1975). The ceU suspensions were mixed with equal volumes of 8% FCS, and ceUs were filtered through Nitex cloth (pore size 28 µ ). Filtered ceUs were sedimented by 1000 g centrifugation, then resuspended in Medium 3. Aliquots of ceU suspension were passed through a 6300 A cytograph (Bio/Physics Systems, Inc., New York, U.S.A.) to determine ceU concentration. CeU viabiUty was estimated by the exclusion of trypan blue (Paul, 1970). A small volume of the cell suspensions was checked histologicaUy after fixation in Bouin and staining with Giemsa. Approximately 5 IO4 ceUs were incubated in 2 ml Medium 3. RadiolabeUed DHT, dissolved in propylene glycol-ethanol (1:1 v/v), was added to each culture dish in a volume of 10 ul. Incubation was stopped by pipetting the ceU suspension into 10 ml ethyl acetate for steroid extractions. Separation ofsteroids Residue of the steroid extract was dissolved in three successive washes of chloroform- methanol (1:1, v/v) and spotted on t.l.c. plates. The solvent (benzene-acetone, 4:1 v/v) front ran 16 cm in each of two successive developments of the plates. The Rr values (steroid migration distance/solvent migration distance) for androstenedione, DHT, testosterone and androstanediol (3a-diol plus 3ß-diol) were 0-85, 0-75, 0-62 and 0-52, respectively. Sections of the experimental lanes corresponding to each standard were marked, cut, and placed into scintiUation vials for counting. The remainder of the experimental lanes was cut into 1 cm strips, placed in scintiUation Downloaded from Bioscientifica.com at 09/28/2021 11:12:05AM via free access vials, and counted. The portion of the U.c. plate containing 3a-diol and 3ß-diol was scraped into conical centrifuge tubes and extracted with chloroform—methanol (1:1 v/v). The extract was dried under nitrogen, dissolved in methanol (20 ul), and half was injected into a high pressure liquid Chromatograph (µ-Bondapak C-18 column: Waters Assoc, Milford, U.S.A.). The 3a-diol and 3ß-diol eluted from the column with retention volumes of 16-5 and 23 ml, respectively, in acetonitrile—water (40/60) at a flow rate of 0-5 ml/min. The eluted compounds were collected in scintillation vials, dried, and counted. Definitive proof of structure of 3a-diol and 3 ß-diol was achieved by recrystallization to constant specific activity. The respective steroids extracted from replicate samples for each incubation were pooled and the 3 epimers were recrystaUized through three solvent systems (Sowell, Folman & Eik-Nes, 1974). Results General CeU viabüity was initiaUy 95-100%. After incubation for 24 h, 80-90% of the floating cells continued to exclude trypan blue. Variable numbers of ceUs were attached to the bottom of the dishes, but were not examined for viabüity. Histological examination of the suspensions revealed ceUs at the expected stages of spermatogenesis (see Clermont & Perey, 1957). Ethyl acetate extraction of DHT, 3a-diol and 3 ß-diol from culture medium recovered 80- 82% labeUed steroid but only 90-95% of this could be spotted on U.c. plates. Nearly aU (97- 99%) the radiolabeUed compounds chromatographed with DHT, 3a- or 3ß-diol; the rest of the radioactivity was randomly distributed on the U.c. lane. Extraction of the diol locus gave 90- 95% of that label, and all this radioactivity was recovered after further extraction and was associated only with 3a-diol and 3ß-diol standards. The specific activity (c.p.m.) of 3a-diol and 3ß-diol synthesized from [3H]DHT by testicular ceUs during incubation remained constant (2080 and 1659; 2042 and 1606; 2073 and 1622 respectively) during recrystaUization through ethanol-water (1:1, v/v), methanol-water (1:1, v/v), and acetone-water (1:1, v/v). Age response Preliminary experiments showed that cultures containing approximately 4 IO4 cells and incubated for 24 h were not substrate-Umited for 3a- and 3ß-diol production when the DHT concentration was 5-10 Hg/ml. For 24-h cultures of testicular ceUs from 15-day-old rats, 10 µ% DHT/ml gave Unear production of 3a- and 3ß-diols (r2 = 0-86), indicating ceU viabiUty. Conversion of DHT (10 ng/ml) was directly related (r2 = 0-99) to cell concentration. The specific activity of the products was assumed to be similar to that of the added DHT.