Regulation by testosterone and insulin of citrate secretion and protein synthesis in explanted mouse prostates.

Ventral prostates taken from orchiectomized mice show no detectable development when cultured for four days on a chemically defined basal medium, but show extensive development if insulin is added to the basal medium.1 In these earlier studies with retrogressed prostates,1' 2 insulin added in vitro enhanced the incorporation of C14-amino acid into protein, increased the incorporation of labeled precursor into ribonucleic acid, and promoted histological repair of cells in the secretory epithelium. In contrast, under these same conditions of culture, testosterone, a potent hormonal stimulant for the prostate in vivo, gave no demonstrable repair of the retrogressed organ in vitro. The present experiments with prostates from orchiectomized donors demonstrate for the first time that testosterone added in vitro can evoke two of its principal physiological effects, namely, augmentation of citrate secretion and stimulation of protein synthesis, provided that insulin also is added to the culture medium. M\Ioreover, testosterone added alone in vitro augments both protein synthesis and citrate secretion in partially repaired prostates. M3faterials and Mlethods.-iiale mice of the Swiss white strain weighing 25-27 gm were orchiectomized at 9 weeks of age; the testis, epididymis, and surrounding fat pad were removed, together with as much of the vas deferens and associated fat as feasible. Three weeks after gonadectomy,' mice were sacrificed by decapitation. Ventral prostates were dissected free of the urethra and placed in culture for 4 days at 360; the gas phase was 5% C02-95% 02. After the first 2 days in culture, explants were transferred to fresh medium and cultured for an additional 48 hr; in experiments designed to assess the effect of the hormones on protein synthesis, this second medium contained DL-leucine-1C14 (0.055 ,c/ml). Cultures were randomized, and each entry in Tables 1-3 is a comI)osite of pooled data from two or three experiments; explants were so distributed that a proportionate number in each group was tested against a similar number in every other group. Culture medium: The basal medium was made up of 4 parts Waymouth's tissue culture medium,4 prepared from stable stock solutions,' and 1 part agar (a freshly autoclaved preparation containing 165 mg agar/10 ml of glass-distilled water). Concentrated solutions of the hormones6 and of leucine-1-C"4 were prepared as follows: DLleucine-1-C14 (1 mg/ml in 0.03 N HCl); insulin (1 mg crystalline zinc insulin/ml in 0.03 N HCI); testosterone (an aqueous suspension, 0.31 mg/ml of buffer solution to which Tween 80 had been added).7 Determination of citrate; prostates partially repaired: Preliminary studies disclosed that the amount of citrate formed by retrogressed prostates during 4 days of cultureeven though two or three glands were pooled-was too small to be measured; hence, only partially repaired organs were used in these studies. Prostates were taken from orchiectomized mice that had been pretreated with androgen; two subcutaneous injections of testosterone lpropionate (500 Ag/O.10 ml in aqueous suspension) were given, one injection at 96-100 hr and the other at 48-52 hr prior to sacrifice. Two prostates were used for each citrate determination. Explants were taken off the medium, placed in homogenizing tubes and quickly frozen by plunging the tubes into a cold ethanol bath to which (Irv