Androgenic stimulation (0.1 microM testosterone or 5 alpha-dihydrotestosterone in the medium) of cultured human epididymal tubules increased the synthesis of five proteins, identified by their mobility relative to albumin (Ra) in polyacrylamide gels as 0.31, 0.43, 0.67, 0.81 and 1.01. This effect was inhibited by the simultaneous presence of 10 microM cyproterone acetate in the medium. The caput epididymidis was the most active region in the production of these proteins and a gradient of decreasing activity was found in successive segments. The appearance of induced proteins in the culture medium suggests their secretory nature, while some data indicate that androgens may also affect the secretory process. Bands corresponding to Ra 0.31, 0.43, 0.68 and 1.01 were found in caput and cauda epididymidis fluids, while bands coincident with Ra 0.31 and 0.43 were consistently found in extracts (0.5 M NaCl) of caudal spermatozoa. Preliminary determinations of molecular weight and isoelectric point for the different bands yielded the following results: Ra 0.31, 38,000 and 5.8; Ra 0.43, 21,000 and 6.2; Ra 0.68, 69,000 and 5.1; Ra 0.81, 13,900 and 6.8; and Ra 1.01, 29,000 and 6.8.

The specific activity of epididymal 5alpha-reductase (pmol 5alpha-reduced products mg protein-1 h-1) decreased by 17, 44, 58 and 83% of the initial value and its total activity (nmol 5alpha-reduced products organ-1 h-1) decreased by 66, 85, 94 and 98% 2, 4, 8 and 14 days respectively, after castration. The loss of total activity always exceeded the decrease in organ weight and protein content. The decline in enzymic activity could be prevented by implantation of testosterone at the time of castration. Administration of testosterone propionate (200 microgram/day) for 12 days starting 1 month after castration was associated with the weights of the accessory organs returning to the control values and although the specific activity of 5alpha-reductase was almost completely restored by this treatment, the total activity of the nuclear fraction remained at 49% of the control value. Recombination experiments demonstrated that the effect of androgens is not mediated by a factor present in the soluble fraction and the concomitant administration of androgen and either cycloheximide or actinomycin D blocked the effect of androgen. These data suggest that androgens stimulate the synthesis of epididymal 5alpha-reductase.

The concentration of dihydrotestosterone was measured by radioimmunoassay in nuclear and cytoplasmic extracts from rat ventral prostates. In the regenerating prostates of castrated rats treated with dihydrotestosterone for 4 days, the nuclear concentration of this steroid increased from approx. 70nM to 800nM as a linear function of the injected dose, whereas the cytoplasmic concentration remained relatively constant (70-130nM). Isotope-exchange measurements of nuclear androgen receptors by using [3H]methyltrienolone indicated that, although the concentration of nuclear dihydrotestosterone was several-fold higher than the concentration of androgen receptors, they were logarithmically related. The recruitment of prostatic cells into the growth fraction and the stimulation of 5 a-reductase activity were more directly correlated to the nuclear concentration of androgen receptors than to the total nuclear concentration of dihydrotestosterone. Maximal restoration of a specific isoenzyme of acid phosphatase was achieved when approx. 2000 androgen receptors were present in the prostatic nuclei; higher concentrations of nuclear androgen receptors were associated with decreased amounts of this enzyme. Hence the results imply, first, that the total amount of dihydrotestosterone accumulated by nuclei is not a direct consequence of carrier-mediated transport by androgen receptors, and, secondly, that, whereas acid phosphatase may be differentially controlled by androgens in the regenerating prostate, increases in the amount of cell proliferation and 5a-reductase activity directly parallel increases in the nuclear concentration of androgen receptors.In the prostate, androgenic regulation of physiological functions and cellular growth is attributed largely to the formation, and subsequent binding to chromatin, of androgen-receptor complexes (King & Mainwaring, 1974 (Coffey, 1974;Rennie et al., 1975;Van Doorn et al., 1976). Although it has been postulated that androgen receptors are involved in the uptake and retention of dihydrotestosterone by target-cell * To whom correspondence should be addressed.Vol. 200 nuclei (Rennie & Bruchovsky, 1973;Bruchovsky et al., 1975a), a direct stoicheiometric relationship has not been demonstrated. When castrated rats are injected with radioactive androgen in the ventral prostate the concentration of radioisotope measured in nuclei exceeds that measured in the cytoplasm (Bruchovsky et al., 1975b). However, the concentration of androgen-receptor complexes recovered from the nuclei is insufficient to account for the nuclear influx of androgens if a mole-to-mole relationship is assumed. Furthermore, radioimmunoassay measurements of dihydrotestosterone in ventral prostates from non-castrated rats have indicated that only about 20% of the endogenous nuclear androgen is bound to androgen receptors (Bruchovsky et al., 1980). Whether the large nuclear concentration of non-receptor-bound dihydrotestosterone is achieved through a receptor mechanism or

Seven biospecific adsorbents containing immobilized androgens were synthesized: dihydrotestosterone-17~-succinyl agarose, and both the unsubstituted and the 17P-acetyl derivatives of dihydrotestosterone-7a-undecanoyl agarose, testosterone-7a-undecanoyl agarose, and 19nortestosterone-7a-undecanoyl agarose. The retention capacities for nuclear androgen receptor were generally between 40-80% with little variation in reproducibility; the amount of binding was greatest with dihydrotestosterone-17fi-succinyl agarose and dihydrotestosterone-17~-acetoxy-7a-undecanoyl agarose. Rapid flow rates were obtained with all gels, and no tendency for decomposition was observed over a period of 1 year. Factors that affected retention included the concentration of immobilized androgen, length of the linker arm, occupation of receptor sites, interval of contact with the gel, and temperature of incubation. Chemical dissociation of androgens from androgen receptor complexes with 0.2 mM mersalyl increased the retention of receptor by dihydrotestosterone-17~-succinyl agarose.Two elutants showed promise for the dissociation of gel-bound receptor: I ) 0.2 m M mersalyl in the presence of 1.5 mg/ml of ovalbumin; 2) 10% (vlv) dimethy1formamide:water containing 30 p M [ 1,2-3H] dihydrotestosterone and 0.5 M sodium thiocyanate. WDe Larminat et al of androgen receptor, namely the rat prostate, are not available in sufficient quantity to expedite purification procedures. Second, where the supply of tissue is plentiful, as in the case of human prostate, the concentration of androgen receptor is low. We have attempted to circumvent these obstacles by developing methods for concentrating nuclear androgen receptor based on the use of micrococcal nuclease [4] and lyophilization of nuclear extracts 151. More recently, we have synthesized a number of matrices with immobilized androgens for affinity chromatography, and in this report we describe the methods for the preparation of the materials and explore their potential for the purification of the nuclear form of androgen receptor.

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