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Experimental Cell Research 96 (1975) 31-36

QUALITATIVE DIFFERENCES IN TESTOSTERONE

M E T A B O L I S M AS A N I N D I C A T I O N O F

CELLULAR HETEROGENEITY

IN FIBROBLAST MONOLAYERS

DERIVED FROM HUMAN PREPUTIAL SKIN M. KAUFMAN, L. PINSKY, 1 C. STRAISFELD, B. SHANFIELD and B. ZILAHI Cell Genetics Laboratory, L a d y Davis Institute f o r Medical Research, Jewish General Hospital, and D e p a r t m e n t s o f Pediatrics and Biology, McGill University, Montreal, Quebec, Canada H 3 T IE2

SUMMARY The metabolism of testosterone by fibroblasts cultured from foreskin of newborns, young children and adults has been studied in monolayers incubated with testosterone-4-[~4C] for 1 to 48 h. Three patterns of testosterone metabolite accumulation were observed; they were not correlated with the age of the foreskin donors. The different patterns of testosterone metabolite accumulation exhibited by the early-passage subcultures of sister strains developed simultaneously from single explants of one prepuce may reflect heritable heterogeneity of fibroblast precursors in situ. This hypothesis is supported by the derivation from primary explant fibroblasts of single-cell clones that retained their distinctive patterns of testosterone metabolism during serial subcultivation to senescence.

It is generally assumed that all serially subculturable fibroblasts derived from a single human skin biopsy have heritably identical phenotypes except for those functional disparities [1, 2] associated with varying replicative potential [3]. The failure to record significant variability among presenescent wild-type clones while searching for skin fibroblast mosaicism in human female carriers of X-linked genes has supported the general assumption, at least in respect to the few enzyme activities surveyed. On the other hand, if some of the fibroblasts occupying different depths of the dermis were heritably specialized, and if some of the differentiated cells in a skin biopsy (adipocytes, endothelium) could adopt a fibro3-751815

blastic morphology in vitro, then considerable metabolic heterogeneity might exist among the cells making up the initial fibroblast monolayer derived from a primary skin explant. On the same basis, one could expect to derive sister strains from a single human skin biopsy that differed in one or more metabolic properties. Furthermore, chance alone or selective factors could explain phenotypic alterations in such sister strains during the course of their respective subculture life histories. Our attention was directed to these important questions during a study on the metabolism of testosterone by fibroblasts cultured from foreskin donated by human subjects of various ages. We had reported Exptl Cell Res 96 (1975)

32

M. Kaufman et al.

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Fig. 1. A s c h e m e of testosterone metabolism. O, T e s t o s t e r o n e ; A, dihydrotestosterone; II, androstanediol; 9 a n d r o s t e n e d i o n e ; /x, androstanedione; IS], a n d r o s t e r o n e . Routes 1 a n d 3 h a v e been labelled [21] the "17fl-hydroxyl" and " 1 7 - k e t o n i c " p a t h w a y s , respectively. Route 2 is hypothetical and is d i s c u s s e d in the caption to fig. 2.

strains developed from adult labium majus accumulated androstenedione (fig. 1) as their major initial metabolite [9]. MATERIALS AND METHODS T h e following trivial n a m e s h a v e b e e n used: Dihydrotestosterone, 17fl-hydroxy-5a-androstan-3-one; A n drostenedione, androst-4-ene-3, 17-dione; A n d r o s t a n e dione, 5c~-androstan-3, 17-dione; A n d r o s t e r o n e , 3~hydroxy-5ct-androstan-17-one; Androstanediol, 5aa n d r o s t a n - 3 a , 17fl-diol and 5a-androstan-3fl, 17fl-diol. Fibroblast strains were derived from prepuce explants as previously described [10]. Sister strains of two classes were generated from individual foreskins. In one class primary explant dishes were fed m e d i u m s u p p l e m e n t e d with 15 % fetal calf s e r u m or 6 % fetal plus 6 % n e w b o r n calf s e r u m (single lots of s e r u m were used throughout). T h e fibroblasts derived in one type of m e d i u m were subcultured in it, t h e r e b y segregating t h e m p e r m a n e n t l y from sister fibroblasts e x p o s e d to the second type o f m e d i u m . N o a t t e m p t was m a d e to segregate fibroblasts derived from individual explant dishes. In contrast, sister strains of the second class were isolated in one type of m e d i u m from individual foreskins simply by assigning one 1 m m 3 explant, c h o s e n at r a n d o m , to each Petri dish. T h e fibroblasts appearing in each dish were t h e n p e r m a n e n t l y segregated from their sister fibroblasts appearing in other dishes. Clones were obtained by plating 250 fibroblasts, rem o v e d directly from primary explant dishes, on 10 cm

earlier [4] that fibroblasts serially subcultured from human genital skin metabolized testosterone faster than fibroblasts derived from non-genital skin of individual Table 1. Donor age and pattern of donors. This accorded with data derived testosterone metabolism o f early-passage from fresh slices of human adult [5] and fibroblast strains derived from prepuce neonatal [6] skin. The rate at which the % D H T and its preputial skin slices convert testosterone derivatives specifically to dihydrotestosterone (DHT) is Strain D o n o r age Pattern (24 h) high during the first 3 months of life, then Newborn 1 90 falls progressively so that in the adult the FOFR R117 Newborn 1 + 2~ 44 c rate approaches that of nongenital skin [7]. K A T Newborn 1 +3 b LXF 8 2 84c With regard to the nature of testosterone D X F Child 1 74 10 2 67 c metabolism carried out by slices of human Y X F LF 11 3 genital skin, it has been observed that new- A SJ-2f 21 2 63 c born foreskin [8] and adult scrotal skin [5] D F F 24 1 77 HF Adult 1 68 produce 17fl-OH (fig. 1) metabolites of FK 115 42 1 77 testosterone predominantly, whereas fetal was a p p r o x i m a t e l y equal u s e of patterns 1 genital skin of both sexes as well as adult aandThere 2. labium majus skin accumulate 17=0 (fig. 1) b There was approximately equal u s e of patterns 1 3. metabolites, predominantly [5]. In accord cand Includes 17=0 metabolites p r e s u m e d to be formed with the last observation, two fibroblast via dihydrotestosterone. Exptl Cell Res 96 (1975)

Testosterone metabolism by preputial fibroblasts

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dishes containing 4 ml of Ham's F-12 medium [11]. To ascertain their single-cell origin, each dish was scanned quickly 24 h after plating and the location of well isolated cells was noted with an ink marker mounted on the objective head of a Zeiss inverted microscope. Healthy clones were subcultured 2 weeks later by trypsinization with the aid of stainless steel cylinders whose lower edges were made adherent by application of silicone grease. To assess testosterone metabolism approx. 10~ cells were planted into each of two 60 mm plastic Petri dishes. When the cultures were confluent the initial medium was removed, the monolayer was washed and 2 ml of experimental medium containing testosterone-4[14C] (57.5 mCi/mM; 0.5/xCi/2 ml medium) was added to each dish. 0.2 ml samples of medium were removed from duplicate dishes 1, 4, 8, 12, 24 and 48 h later, and were extracted immediately with chloroform. Testosterone and its metabolites (dihydrotestosterone, androstenedione, androstanedione, androsterone, androstanediol) were isolated from the medium samples as described fully elsewhere [12].

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radioactivity. Patterns of testosterone metabolite accumulation. The symbols are defined in the caption to fig. 1. Each panel depicts the results of a single experiment representing numerous cultures with the same pattern of metabolism. Each point is the mean at least of duplicates. Variation of replicates seldom exceeded 5 %. (a) Type 1 pattern of testosterone metabolism. The accumulation of dihydrotestosterone is followed chiefly by that of androstanediol. This pattern is inferred to represent the "17fl-hydroxyl" pathway of testosterone metabolism shown as route 1 in fig. 1; (b) type 3 pattern of testosterone metabolism. The accumulation of androstenedione is followed successively by that of androstanedione and androsterone. This pattern is inferred to represent the "17-ketonic" pathway of testosterone metabolism shown as route 3 in fig. 1; (c) type 2 (crossover) pattern of testosterone metabolism. The accumulation of dihydrotestosterone, a 17/3hydroxyl compound, is followed by the successive

One can recognize three distinct patterns of testosterone metabolite accumulation in early-passage fibroblast monolayers derived from human foreskin (fig. 2a, c). Some cultures use one of the three patterns predominantly, while others appear to use two of them about equally. Neither the pattern of testosterone metabolism of a culture nor the rate at which it produces DHT and its presumed derivatives are correlated with the age of the foreskin donor (table 1). While accumulating these initial results, we observed that early-passage sister cultures could metabolize testosterone differently without relation to the different serum supplements in their media. To assess the suggestion that such sister cultures were heritably different, sister strains were systematically developed from a single foreskin in one type of medium by placing one 1

accumulation of two 17-ketonic derivatives, androstanedione and androsterone. This pattern is postulated to represent a pathway of testosterone metabolism shown as route 2 in fig. 1. Exptl Cell Res 96 (1975)

34

M. Kaufman et al.

mm 3 explant into each of numerous Petri dishes, and then permanently segregating the fibroblasts derived from each dish. The results in table 2 indicate that such sister strains could also exhibit different patterns of testosterone metabolism in monolayers derived either directly from primary explant dishes or after a few subcultures at low split ratios. To persue the hypothesis that such sister-strain differences might reflect heritable heterogeneity of individual fibroblasts, the fibroblasts emanating from a single prepuce explant were cloned. Differences in testosterone metabolism among the clones were observed in their earliest subcultures, and persisted through successive subcultures until senescence (table 3). DISCUSSION The recognition of different patterns of testosterone metabolism in the earliest subcultures of sister strains developed from single explants of one prepuce was surprising. This observation was postulated to re-

Table 2. Pattern of testosterone metabolism in very early-passage fibroblast monolayers representing sister strains developed from individual foreskins Subject (years)

Sister-strain

Pattern

P E F (8)

3 4 7 10 11 14 1 2 3 5 6 8 10

3 2 2 1+2 2 3 1+2 1+2 3 1 1 1+3 3

TOF (3)

Exptl Cell Res 96 (1975)

Table 3. Patterns of testosterone metabolite accumulation in clones derived from primary preputial skin fibroblasts Pattern

No. of clones

1 3 1+3

8 4 4

flect heritable cellular differences of the strains because it could not be explained by any of the following conditions of the specific cultures assayed: (1) their mean population doubling level (in vitro age); (2) their confluent population density or mitotic index; (3) their rate of testosterone metabolism; and (4) such environmental variables as pH and temperature. The isolation from primary preputial fibroblasts of single-cell clones with distinctive patterns of testosterone metabolism strengthens the hypothesis. Heterogeneity of sister strains cultured from a single foreskin has also been observed by Milunsky et al. [13]. They found variations of 60-500% in various lysosomal acid hydrolase activities in the earliest subcultures of identically-handled strains, and suggested that heritable fibroblast heterogeneity could account for these strain differences. Wilson & Walker [7] found that the mucinous connective tissue between the two layers of skin in prepuce had a five-fold greater ability to convert testosterone to D H T than did samples of equal wet weight from dermis, but they could not attribute this difference to a disparity between the cells inhabiting these two layers of prepuce. Our results [14] and those of Milunsky et al. [13] clearly favor this interpretation. It is known that cells derived from different human organs may adopt a fibroblastic

Testosterone metabolism by preputial fibroblasts morphology in monolayer culture while retaining organ-specificity. For instance, human fetal fibroblasts from lung display a sex difference in glucose-6-phosphate dehydrogenase (G6PD) activity, while those from skin do not [15]. It is often unclear, however, whether such 'fibroblasts' represent connective tissue cellular precursors in the organ or parenchymal cells with altered morphology in vitro. It is not well known that human skin explants can yield serially subculturable fibroblasts which reflect their site of origin. Thus, G6PD activity is greater in fibroblasts from newborn foreskin than in those from non-genital skin provided by donors of various ages [16]. Similarly, fibroblasts serially subcultured from prepuce or labium majus metabolise testosterone faster (regardless of pattern) than fibroblasts derived simultaneously from non-genital skin of individual donors [4]. The proposal that fibroblasts derived from any one skin site may differ heritably (in ways not related to their varying replicative potentials) is only one step removed from the fact that different skin sites may yield different kinds of fibroblasts. Indeed, different age-determined proportions of heritably distinct fibroblasts at any one site may explain the glycolytic enzyme differences noted among skin fibroblasts derived from donors of different ages [16, 17]. It is noteworthy, in contrast, that cultured fibroblasts did not reflect age-related differences in the ability of preputial skin slices to convert testosterone to dihydrotestosterone [7]. The basis for the preputial fibroblast heterogeneity uncovered by the present work is unknown: variation in the apoenzymes responsible for testosterone metabolism may be at fault, or varying levels of their co-factors may be involved. The presence or absence of cytosol

35

androgen receptors is not a critical variable, since we have observed different patterns of testosterone metabolism in sister strains when replicate monolayers of each have been found to have such receptors in equal amounts and with the same binding properties [18]. The genetic form of human incomplete male pseudohermaphroditism which is determined by homozygosity for an autosomal recessive gene [19] has recently been attributed to a primary deficiency of the enzyme responsible for converting testosterone to D H T in the urogenital sinus and the external genital primordia, including that of the prepuce [20]. Our results suggest that it may be impossible to define a reliable phenotype of this mutation in unselected fibroblast monolayers derived from the prepuce of such patients compared with controls. This work was supported by a grant (MT-2830) from the Medical Research Council of Canada.

REFERENCES 1. Houck, J C, Sharma, V K & Hayflick, L, Proc soc exp biol med 137 (1971) 331. 2. Singal, D P & Goldstein, S, J clin invest 52 (1973) 2259. 3. Smith, J R & Hayflick, L, J cell biol 62 (1974) 48. 4. Pinsky, L, Finkelberg, R, Straisfeld, C, Zilahi, B, Kaufman, M & Hall, G, Biochem biophys res commun 46 (1972) 364. 5. Flamigni, C, Collins, W, P, Koullapis, E N, Craft, I, Dewhurst, C J & Sommerville, I F, J clin endocrinol metab 32 (1971) 737. 6. Gomez, E C & Hsia, S L, Biochemistry 7 (1968) 24. 7. Wilson, J D & Walker, J D, J clin invest 48 (1969) 371. 8. Gomez, E C, Hsia, S L & Frost, P, J clin endocrinol metab 34 (1972) 417. 9. Mulay, S, Finkelberg, R, Pinsky, L & Solomon, S, J clin endocrinol metab 34 (1972) 133. 10. Pinsky, L, Miller, J, Shanfield, B, Watters, G & Wolfe, L S, A m j human genet 26 (1974) 563. 11. Ham, R G, Proc natl acad sci US 52 (1965) 288. 12. Pinsky, L, Kaufman, M, Straisfeld, C & Shanfield, B, J clin endocrinol metab 39 (1974) 395. 13. Milunsky, A, Spielvogel, C & Kanfer, J N, Life sci 11 (1972) 1107. Exptl Cell Res 96 (1975)

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et al.

14. Kaufman, M, Straisfeld, C, Shanfield, B] Zilahi, B & Pinsky, L, A m j human genet 26 (1974) 47A. 15. Steele, M W & Migeon, B R, Biochem genet 9 (1973) 163. 16. Steele, M W & Owens, K W, Biochem genet 9 (1973) 147. 17. Condon, M A, Oski, F, Dimauro, S & Mellman, W, Nature new biol 229 (1971) 214. 18. Kaufman, M & Pinsky, L, Clin genet. In press.

Exptl Cell Res 96 (1975)

19. Opitz, J M, Simpson, J L, Sarto, G E, Summit, R L, New, M & German, J, Clin genet 3 (1972) 1. 20. Walsh, P C, Madden, J D, Harrod, M J, Goldstein, J L, MacDonald, P C & Wilson, J D, New eng j med 291 (1974) 944. 21. Beaulieu, E-E & Mauvais-Jarvis, P, J biol chem 239 (1964) 1569. Received April 15, 1975