At short intervals after the intravenous administration of oestradiol-17beta, diethylstilboestrol, testosterone or saline control solution to ovariectomized rats, highly purified lysosome samples were prepared in substantial yield from preputial glands, sex accessory organs rich in these organelles. The preparations were essentially devoid of mitochondrial contamination. Exposure in vivo to doses of these hormones varying from 0.1 to 5mug/100g body wt. provoked dose-dependent labilization of the lysosomal membrane surface, as evidenced by significantly diminished structural latency of several characteristic acid hydrolases, including acid phosphatase, beta-glucuronidase and acid ribonuclease II, when such preparations were subsequently challenged in vitro with autolytic conditions, detergent or mechanical stress. Enhanced lytic susceptibility induced by hormone pretreatment was occasionally detectable in the initial preparation without further provocative stimuli in vitro. Comparable results were obtained with the corresponding fractions of uterus, despite the more limited concentration of lysosomes in this steroidal target organ. By the present criteria oestradiol-17alpha was essentially inert, even in a dose 25 times that effective for its active beta-epimer (<0.1mug/100g body wt.). Pretreatment with diethylstilboestrol exerted substantial membrane-destabilizing influence in preputial-gland lysosome samples from orchidectomized rats. Moreover, administration of testosterone to gonadectomized animals resulted in essentially equivalent dose-dependent augmentation of lysosomal enzyme release in preputial-gland preparations of either sex. The membrane stability of lysosome-enriched preparations from uterus, on the other hand, was unaffected by testosterone pretreatment. The sensitivity, specificity and selectivity of the lysosomal response to sex steroids provide evidence for the physiological significance of this phenomenon as a general mechanism for mediation of secondary biochemical transformations in the hormone-stimulated target cell.

Recent evidence from these laboratories has implicated the primary lysosomal population of specific target cells in the selective reception of steroid hormones at the cell periphery. This phenomenon was shown to be associated with covert labilization of the lysosomal bounding membrane. It has now been demonstrated that this interaction with trophic hormones results in rapid migration of lysosomes to a perinuclear position. Suspensions of nuclei were isolated from preputial glands of ovariectomized rats within short intervals after administration of gonadal hormone in vivo. Identification of cellular organelles by ultraviolet fluorescence microscopy was carried out on preparations subjected to intravital staining in vitro with acridine orange. Lysosomes were present in high concentrations in nuclear fractions of preputial glands from animals pretreated with oestradiol-17\g=b\, diethylstilboestrol, or testosterone, and essentially absent in preparations obtained from animals injected with oestradiol-17\g=a\or control solution. The response could be detected within 1 min of hormone administration, was obtained with as little as 0\m=.\1\g=m\ghormone/100 g body wt, and was not observed in preparations from lung tissue. In the early stages of hormonal activation, lysosomes in preputial gland preparations formed adherent perinuclear clusters and appeared relatively intact. However, lysosome fragmentation and dissolution rapidly ensued and was accompanied by the appearance of polynuclear\p=m-\multilysosomalaggregates. Concomitantly, isolated lysosome preparations subjected to enzymatic analyses revealed increased release from the particle-bound form of hydrolytic enzymes characteristic of these organelles. Progressive nuclear metachromasy was also observed after administration of the active hormones, suggesting the breakdown of macromolecules which yield products with enhanced capacity for binding acridine orange. These results suggested that modification of the nuclear surface and of intranuclear constituents, leading to altered adhesive-* Some of the present data were presented in preliminary form at the Third International Congress on Hormonal Steroids,

Analysis of the properties of the predominant acid proteinase of rat preputial gland has been carried out on partially purified extracts of lysosomes isolated from this organ, using a fluorometric assay and the synthetic substrate, carbobenzyloxy-alanyl-arginyl-arginyl-4-methoxy-~-naphthylamide. By means of exclusion chromatography on Sephadex (3-200, a molecular weight of =z 25000 was estimated. The enzymic activity exhibited a pH optimum of 6.2. Calcium ion was required for full activity within a narrow range of concentrations, beyond which inhibition was noted. Substrate specificity of the enzyme toward arginyl and lysyl residues and an absolute requirement for sulfhydryl activation, coupled with the capacity to inactivate aldolase, strongly suggested cathepsin B1-like character. This inference was supported by relative efficiencies of several classes of inhibitors. Powerful constraint of activity was exhibited by the known cathepsin-B 1 inhibitors, leupeptin and antipain, while pepstatin, a recognized antagonist of cathepsin D, was without influence. Neither lima bean trypsin inhibitor nor phenylmethylsulfonyl fluoride was active. However, substantial inhibition was seen with tosyl-L-lysyl-chloromethyl ketone, iodoacetate, and ovomucoid. Collectively, these data strongly implicate cathepsin B 1 as the predominant lysosomal proteinase of rat preputialgland. In extension of previous work with selected lysosomal hydrolases, estradiol-l7P, but not the inactive 17a-epimer, elicited, within 2 min of its administration, a marked reduction in structural latency of cathepsin B1 in lysosome-enriched fractions isolated from the preputial gland of the ovariectomized rat, without evoking concomitant alteration in the total activities in the corresponding unfractionated homogenates. Moreover, administration of the active hormone led to the rapid appearance of this enzymic activity, foreign to the nucleus in the controls, in ultrapurified nuclei isolated from this organ. The potential significance of such recompartmentation in the subsequent secondary responses initiated by the hormone, including mitogenesis, is discussed.Available evidence implicates gonadal steroids

The present investigation was designed to determine whether the rapid intracellular redistribution of lysosomes to a perinuclear position in reproductive target cells after administration of specific gonadal hormones in vivo was accompanied by evidence of penetration of the nucleoplasm by lysosomal marker enzymes. Nuclei were prepared by conventional isolation and purification procedures from preputial glands or uteri that were excised from gonadectornized rats within 2-15 min after intravenous injection of physiological doses of estradiol-17/3, testosterone or saline control solutions. Control and experimental preparations were essentially "pure", as judged by routine phasecontrast microscopic criteria in general use. However, nuclei from target cells of animals given hormone contained substantial activities of representative lysosomal hydrolases, including acid phosphatase, /3-glucuronidase and acid ribonuclease 11. In contrast, the samples originating from the target tissue of saline-treated controls or from non-target tissues contained minimal concentrations of these enzyme activities. After further purification of the nuclei by removal of the outer nuclear membrane through brief exposure to very low concentrations of Triton X-100 in the cold, the resultant 'ultrapurified' nuclei retained significant concentrations of structurally latent lysosomal marker enzymes after hormonal pretreatment without evidence of appreciable contamination by enzymes of mitochondria1 origin. Similar results were obtained with nuclei isolated by non-aqueous procedures. These combined observations appear to exclude adsorption artifacts. The total activities of lysosomal enzymes in the unfractionated homogenates and the MgZ+-dependent ribonuclease activity indigenous to the nuclear compartment were unaffected by prior hormone treatment. These results are consistent with the hypothesis that invasion of the nucleoplasm of specific target cells by lysosomal products may serve as a mechanism for triggering genic derepression in steroid hormone-induced growth.

SUMMARY Mating and impregnation were not prevented by adrenalectomy in female rats; they carried their young to term, large litters of viable young were born and lactation was sufficient to maintain the young throughout the usual nursing period. By comparison with sham-operated mother rats, the adrenalectomized mothers gave birth to smaller litters, their pups weighed less and had increased levels of plasma corticosterone. Prenatal and postnatal effects on the offspring were separated by crossfostering procedures. At 21 days of age there was no significant difference among the groups in the number of pups surviving; however, the pups weighed less if the biological or foster mother was adrenalectomized. These differences were less marked in multiparous mothers.