Robert A. Huseby

Organization of androgen biosynthesis in the testis.

Abstract Factors involved in migration of pregnenolone and progesterone from the mitochondria to the endoplasmic reticulum, and in the organization of enzymes involved in androgen biosynthesis in the reticulum have been studied. Pregnenolone and progesterone were differentially concentrated in the endoplasmic vesicle by systems with high capacity and low K a compared with target organ receptors. The differential binding was reduced by mild trypsin treatment or phospholipase C but only slightly by phospholipase A. Under similar conditions all three enzymes markedly reduced 3β- and 17β-dehydrogenases; 17-αhydroxylase and 17,20-lyase activities fell to unmeasurable levels. This was associated with the transfer of NADPH-cyt c reductase activity from the microsomal pellet to the cytosol. A study of the 17-α-hydroxylase and 17,20-lyase reactions using [ 3 H]-progesterone and [ 14 C]-17hydroxyprogesterone indicated that freely diffusible 17-hydroxyprogesterone was not an obligatory intermediate but that the two steps involved consecutive hydroxylation type transfers to an enzymebound intermediate. The overall picture is that of pregnenolone being concentrated and undergoing conversion to progesterone at the outer microsomal surface, progesterone then associating with a cyt-P-450 complex within the membrane which catalyzes consecutive mixed function oxidase reactions, after which the androstenedione formed diffuses outward, partially undergoing 17β-reduction at the outer surface.

Molecular nature of interaction of steroids with biomembranes related to androgen biosynthesis

Abstract Pregnenolone and progesterone were found to be differentially concentrated in the membrane fractions from Leydig cell tumors and cryptorchid testes when equilibrium between membranes and the steroid containing buffer was achieved without metabolism. On the other hand, androstenedione was not significantly bound to any of the membranes. This concentrating system for pregnenolone and progesterone had high capacity and a low association constant. The amounts of pregnenolone bound to the mitochondria-rich fraction (MRF), smooth endoplasmic reticulum (SER) and rough endoplasmic reticulum (RER) were 4.8, 9.9 and 7.3 nmol/mg protein, respectively, when these fractions were incubated with 5 nmol of pregnenolone in 5 ml of the buffer. These differences, however, almost disappeared when the binding data were viewed in relation to the amount of membraneous phospholipid; that is, 0.3 for MRF, 0.29 for SER and 0.34 nmol/μg Pi of phospholipid for RER. The binding of pregnenolone to 3β-ol-dehydrogenase would be negligible, since tumor membranes which showed considerable variation in this enzymatic activity had a fairly constant capacity of the pregnenolone binding. Progesterone is bound to these fractions in a similar manner to pregnenolone. These results might suggest that the steroid-bio-membrane interaction occurs almost exclusively with phospholipids in membranes. The partition coefficients between the phospholipid in SER and the aqueous suspending medium were calculated to be 1.5 × 103 for pregnenolone, 2 × 102 for progesterone and 4.5 for androstenedione.

Regression of an adrenal cortical carcinoma and its neovascular bed following mitotane therapy: A case report

A 48‐year‐old Caucasian woman who had increasing hirsutism over a 2‐year period was finally hospitalized because of acute abdominal pain. Pulmonary metastases were present. An exploratory laparotomy revealed a large, highly vascular, inoperable adrenal neoplasm. Seventeen ketosteroids and 17 ketogenic steroids were significantly elevated. An abdominal aortogram with selective catheterization established the size and vascularity of the abdominal mass. Treatment with mitotane resulted in a remarkably prompt return to normal of the urinary ketosteroids and corticosteroids and regression of the pulmonary metastases. Repeat arteriographic studies showed a marked reduction in the abnormal vascularity as well as in the size of the abdominal mass. The patient has been maintained on mitotane for most of the 4 years since starting therapy. To date there has been no evidence of reactivation of any of the neoplastic foci.

Inverse Changes of Serum Glucuronidase and Esterase of Breast Cancer Patients on Estrogen Therapy.

Summary Simultaneous sera glucuronidase, and esterase determinations have been carried out on the blood samples of 37 post-age 50 healthy control subjects (17 males and 20 females), 11 patients with advanced breast cancer and not on hormone therapy, and on 12 patients with advanced breast cancer receiving estrogen therapy. It was found that the control male subjects had both a higher serum glucuronidase and serum esterase than did the control female subjects. An inverse relationship was found for the changes in sera activity levels of glucuronidase and esterase in the patients with breast cancer subjected to variations in estrogen therapy.

Estrogen-Androgen Antagonism Histology of Mammary Glands and Vaginal Grafts of Male Mice Receiving Estrogens.

Summary 1. Studies of the effect of injections of steroid and non-steroid estrogenic substances on the mammary gland and vaginal mucosa of castrate and non-castrate male mice bearing subcutaneous vaginal grafts are reported. 2. It is suggested that antagonistic actions exist between the naturally-occurring estrogens (estrone, estradiol) and testicular androgens in regard to the mammary gland and vaginal mucosa. 3. Such antagonism did not seem to occur when the synthetic estrogenic substance diethylstilbestrol was employed.

Incidence of mammary tumors in castrate and non-castrate male mice bearing ovarian grafts.

Conclusions Castrate male mice of the F1 hybrid generation between the A and Z stocks bearing subcutaneous ovarian grafts developed mammary cancer more readily than do virgin female mice of this cross. Only 1 of 39 non-castrate males bearing ovarian grafts develop mammary carcinoma as little mammary development occurred. Additional experiments seemed to indicate that the ovaries in the non-castrate male mice were producing an appreciable amount of estrogen although measurably less than those carried in castrate males. There appeared, also, to be an antagonism between estrogens and androgens as far as stimulation of the vaginal and mammary epithelium is concerned. Granulosa cell tumors occasionally occurred in ovarian grafts of old male and female mice of this hybrid cross.

Dehydrogenation of Reduced Pyridine Nucleotides by Leydig Cell Tumors of the Rat Testis

Summary The activities of the cytochrome c reductases and of the D-T diaphorase in rat Leydig cell tumors have been described. The increase in enzymatic activity of the NADH cytochrome c reductase activity in functional tumors derived from interstitial cells of the rat testis is interpreted as being possibly related to hydroxylation of steroids by the neoplastic cells. Meanwhile, the increase in the activity of the D-T diaphorase in the other tumor is interpreted as being an anaplerotic reaction to substitute for the deficient shuttles for the transfer of reducing equivalents from the cytoplasm to the mitochondria observed in tumors.

Successful growth of tumor allografts following explantation, effect of various culture conditions.

Summary Alteration in the antigenicity of a Leydig cell tumor following short-term maintenance in organ culture and manifested by progressive growth of the tumor in allogeneic hosts occurs following incubation in media independent of the following factors: (i) presence or absence of antibiotics in the medium, (ii) incubation at 32 or 37°, (iii) incubation in 5% CO2 in air or in oxygen, and (iv) presence or absence of 10% fetal calf serum in the medium. The frequency of successful allografts is greater in tumors explanted for 6 days than for those cultured for 1 or 2 days. The alteration occurred more frequently after maintenance in Hanks’Balanced Salt Solution and Waymouth Medium than it did after maintenance in medium 199. The occurrence of in vitro-induced alteration in tumor specificity is discussed in relation to other conditions known to alter immunologic specificity and the possible relationship of these to the phenomenon of immunologic enhancement is suggested.

Hydroxybenzene Compounds as Cytoplasmic Fixatives.

Summary Methods for the fixation of the cell cytoplasm employing pyrogallol or resorcinol in neutralized formaldehyde solution are outlined. Tissue pieces fixed with these solutions have, in general, shown few signs of distortion, and the protein cytoplasmic elements of the cells have appeared well preserved, such structures as mitochondria, secretion granules, and some of the specific types of granulation being well maintained. The advantages of these methods lie in the simplicity and rapidity of the procedures, the ease with which the tissue can be sectioned at a thickness of 1 to 2 micra, and the generally satisfactory results obtained.

Influence of age and mammary development upon mammary carcinogenesis following injection of milk agent.

Summary Employing male mice, transplanting ovaries and injecting active milk agent virus at different ages, an experiment was carried out to specifically test the relationship of the state of mammary glad development at the time of the introduction of the milk agent to mammary carcinogenesis. The result would indicate that the relative non-susceptibility of older mice is not due to greater mammary gland development at the time of virus infection.