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Biomedical subjects

R A Huseby

Publications and source records attributed to R A Huseby.

17 recordsLinked to original sources
PubMed1979

The testicular estrogen receptor system in two strains of mice differing in susceptibility to estrogen-induced Leydig cell tumors.

Cytosol obtained from cryptorchid testes of tumor-susceptible BALB/c and resistant C3HBi (Z) mice both bound 17 beta-estradiol (E2) and diethylstilbestrol (DES) specifically. The dissociation constant (Kd) of this binding component (RE) for E2 was determined to approximate 5 x 10(-9) M. Gel filtration of cytosols resulted in a significant increase in the binding constant (Kd approximately 3 x 10(-10) M) with the majority of the complex migrating in the 7-8S area after sucrose gradient centrifugation. Incubation of either untreated or gel-filtered cytosol with [3H]DES resulted in considerable nonspecific binding appearing in the 4S region in a low salt sucrose gradient. This 4S binding of [3H]DES was not inhibited by the addition to the incubation mixtures of a 100-fold excess of either E2 or DES, while the lesser peak at 7-8S as well as the major 7-8S peak formed with E2 were inhibited by both. In vitro translocation of the cytosol RE to the nucleus was demonstrated in both mouse strains using either estrogen. Quantitation of the in vivo translocation, employing the exchange method after a single injection of 2.5 micrograms E2/mouse, revealed a rapid increase in cytoplasmic receptor content accompanied by a concomitant increase in nuclear receptor content. Greater nuclear receptor content was identified in nuclei from BALB/c mice than in those from Z animals 45 min after injection of E2. The binding behavior of E2-RE complexes to nuclei was studied by the KCl extraction method. The percent extracted from the nuclei in the Z strain was significantly greater than that in the BALB/c at all concentrations of KCl tested. Essentially 100% of the RE was extracted from nuclei of Z animals at 0.4 M KCl, while nuclei of BALB/c mice retained 35-40% even in 2 M KCl. Cross-over experiments in a cell-free system suggested that the difference in binding was due to differences in chromatins rather than in nuclear estrogen-receptor complexes. The greater nuclear receptor content and stronger binding of nuclear receptor to chromatin might explain why estrogen-induced phenomena, including neoplastic transformation occur to a much greater degree in the BALB/c strain than in the Z strain of the mouse.

Animals↗
PubMed1978

Evidence of a small molecule in mouse Leydig cell tumors which inhibits the conversion of estrogen receptor from 4S to 5S.

The activation process of cytosol estrogen receptor (RE) from mouse Leydig cell tumors was investigated. When RE from tumors which were affected in their growth by estrogen was heated at 25 C for 30 min in high ionic strength buffer it was converted from native 4.0S to a transformed (5.3S) state. Dialysis of the cytosol from these tumors at 0-4 C in either the presence or absence of estradiol also resulted in conversion to 5.3S when estradiol was added before the sample was applied to the sucrose density gradient. When the dialyzed cytosol was applied to a sucrose density gradient without estradiol, the peak of estrogen binding capacity tested by adding E2 to the fractions from the gradient, occurred at approximately 4S. The dialysate of cytosol had an inhibitory effect on activation by warming. Dialysis of cytosol in the presence of estradiol enhanced the translocation of estradiol-receptor complex to nuclei. Molecular weight (approximately 128,000) and frictional coefficient (f/f0 1.62) of the estrogen-receptor complex dialyzed in the presence of high salt concentration were identical with those of warmed RE complex, but quite different from the native RE (approximately 73,000 daltons, frictional coefficient 1.48). This evidence indicates the presence in the cytosol of one or more small molecules which, when noncovalently bound to RE, inhibit the conversion of the native cytosol RE to the transformed state R'E2RE, having an additional protein unit.

Animals↗
PubMed1978

The possible roles of membrane organization in the activity of androgen biosynthetic enzymes associated with normal or tumorous mouse Leydig cell microsomes.

The enzymes involved in conversion of pregnenolone to testosterone in Leydig cell tumors showed a wide distribution among smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER), and cytosol, while these enzymatic activities in normal testes were associated primarily with smooth endoplasmic reticulum. Progesterone, used as a substrate in the presence of an NADPH-generating system, was metabolized to androstenedione and finally to testosterone by microsomes from some strains of tumor which did not form testosterone from exogenous labeled androstenedione. Treatment of microsomal membranes from normal testes with 0.1 M Ca++ and Mg++ caused a marked decrease in 17 beta-dehydrogenase activity, measured as conversion of exogenous [3H]androstenedione to [3H]-testosterone, without serious effects on activities of 3 beta-ol-dehydrogenase or 17 alpha-hydroxylase. Studies of initial velocity kinetics showed that treatment with magnesium ion resulted in a marked reduction in affinity of androstenedione for 17 beta-dehydrogenase while the maximum velocity was the same as in untreated microsomes. Also, experiments using [14C]progesterone and [3H]androstenedione simultaneously as substrates demonstrated that treatment with Mg++ ion made it more difficult for exogenous [3H]androstenedione to reach the active site of 17 beta-ol-dehydrogenase than [14C]androstenedione formed in the microsomal membrane from [14C]progesterone. Microsomal proteins were more easily solubilized and 3 beta-ol-dehydrogenase was more severely influenced by Mg++ ion in tumor membranes than in normal microsomes.

17-Hydroxysteroid Dehydrogenases↗
PubMed1978

DNA synthesis and DNA polymerase activity in Leydig cells of diethylstilbestrol-stimulated mouse testes.

Using a modification of the collagenase dispersion method of Dufau et al., we examined changes in DNA synthesis produced by estrogens in the interstitial cells of mice that develop malignant Leydig cell tumors after prolonged estrogen administration. Previous work in cryptorchid mice indicated that during continuous estrogen administration [3H]thymidine incorporation into DNA rises to a maximum in 3 to 4 days and then falls to approximately base levels within 2 to 3 weeks. This was confirmed both in Leydig cell concentrates of estrogen-treated mice after either injection with [3H]thymidine or incubation with [3H]thymidine in vitro. This DNA synthesis was blocked by hydroxyurea. DNA synthesis in cells of estrogen-treated BALB/c mice of the Huseby substrain, which have a high incidence of Leydig cell tumors, was 5 to 11 times that in untreated controls. Cells from estrogen-treated C3H/Bi mice, which have a low incidence of Leydig cell tumors, showed only a 2- to 3-fold increase. In the Huseby substrain the rise of DNA synthesis is a peak and subsequent recession were paralleled by a rise and fall in DNA polymerase alpha activity. DNA polymerase beta did not show this variation. In C3H/Bi mice, neither polymerase showed significant change. The evidence suggests that the early estrogen-stimulated DNA synthesis is probably replicative and is associated with increased DNA polymerase alpha activity.

Animals↗
PubMed1977

Lack of influence of hypophysectomy on estrogen-induced DNA synthesis in Leydig cells of BALB/c mice.

In mice of strains susceptible to Leydig cell tumor induction, treatment with estrogens induced a "spurt" of DNA synthesis within the first few days. This synthetic activity generally subsided, until areas of Leydig cell hyperplasia developed several months later. Autoradiographic and quantitative biochemical studies indicated that in BALB/c mice this initial DNA synthetic activity occurred in the absence of the hypophysis and apparently was the result of effects of estrogen directly on Leydig cells. Although hypophysectomy inhibited sperm maturation, [3H]thymidine incorporation into spermatogonia was reduced only slightly 2 weeks after surgery, as was the induced DNA spurt in the interstitial tissues.

Animals↗
PubMed1976

Effects of trypsin and phospholipases A2 and C on enzyme organization in testis microsomes.

Pregnenolone and progesterone concentrated in the microsomal fraction of cryptorchid mouse testis compared with mitochondria and cytosol. While the concentrating mechanisms had high capacity and low association constants the effect did not seem to be due to nonspecific solubility in the lipid components since 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione and testosterone did not show differential concentration. Also digestion with phospholipases A2 and C to the point where most of the phospholipids were specifically split, only lowered the differential binding of pregnenolone and progesterone by less than half. Trypsin had a greater effect, short digestion at 0 degrees C lowering the specific binding to 35-40% and decreasing the steroid dehydrogenases to a similar extent. The members of the mixed function oxidase system in the testis microsomes were particularly sensitive to trypsin, cytochrome P-450 and, as a consequence, 17alpha-hydroxylase and 17, 20-lyase activity being eliminated under tha same conditions while liver microsomal cytochrome P-450 was hardly affected. Bonds split by trypsin seem to play a more important role in the hydroxylase activity of testis microsomes than in the hepatic system.

Androstenedione↗
PubMed1976

Dehydrogenation of reduced pyridine nucleotides by Leydig cell tumors of the rat testis.

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.

Animals↗
PubMed1976

Estrogen-induced Leydig cell tumor in the mouse: a model system for the study of carcinogenesis and hormone dependency.

Malignant neoplasms of endocrine tissues represent almost half of the cancers diagnosed clinically in the United States, and many of these respond to hormonal therapies. Estrogen-induced testicular Leydig cell tumors in the mouse would seem to represent a realistic model for the laboratory investigation of this significant group of cancers. Data accumulated over the past few years clearly show that the Leydig cell is a target tissue for estrogens. Administering large doses of estrogen results in a reduction of enzymes converting progesterone to testosterone and induces a transient, but quantitatively very significant, synthesis of DNA in the Leydig cells of tumor-susceptible strains of mice. Neither of these actions of estrogen is mediated via the hypophysis. It has been demonstrated that the Leydig cells have specific protein receptors in their cytoplasm that bind estrogens and transport them to the nucleus where they are also bound. The genetic composition of the Leydig cells themselves is extremely important for the development of tumors. An adequately functioning pituitary gland is also essential for tumor formation. Confining the testes to the abdomen results in enzyme changes similar to those produced by estrogen administration and significantly augments the development of Leydig cell tumors. Once tumors form they frequently are dependent for their continued growth on estrogenic stimulation and/or on a functioning hypothysis. Regressed tumors may remain dormant for many months only to resume progressive growth when placed in and adequate hormone environment.

Animals↗
PubMed1976

The specificity of gonadotropin binding by the human corpus luteum.

The specificity of gonadotropin binding was studied in fresh and frozen human corpora lutea. Ovine, bovine, and porcine luteinizing hormone (LH) competed with 125I-labeled human LH (125I-hLH) and 125I-labeled human chorionic gonadotropin (125I-hCG) for binding to tissue receptors in homogenates of human corpora lutea frozen for 3 to 12 months. In contrast, oLH, bLH, and pLH competed minimally for 125I-hLH and 125I-hCG binding sites in homogenates of fresh human corpora lutea. Ovine follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) did not compete in homogenates of fresh or frozen tissue. Competition of oLH and hCG for 125I-hCG binding sites at several dose levels in a homogenate of a fresh corpus luteum was studied. One hundred micrograms of oLH and ten nanograms of hCG gave an equivalent competition--a 10,000-fold difference in competitive potency. Only hCG competed with 125I-hCG for binding when the competition of oLH, bLH, pLH, oFSH, oTSH, hCG and hCG subunits, and hCG were compared at the 10-mug level in a homogenate of fresh human corpus luteum. The binding of 125I-labeled homologous human hormones by the corpus luteum was examined in a limited fashion. 125I-Prolactin did not bind to preparations of fresh stroma from a patient with polycystic ovaries nor did it bind to three separate preparations of fresh corpora luteum which did bind 125I-hCG. 125I-hTSH did not show significant binding to a fresh human corpus luteum preparation which did bind 125I-hCG. These studies indicate that the gonadotropin receptor of the fresh human corpus luteum possesses a unique species specificity and illustrate the importance of working with human corpora lutea in their most native state.

Binding, Competitive↗
PubMed1975

Changes in fine structure accompanying estrogen-induced tumorigenesis of Leydig cells in the mouse testis.

The development of estrogen-induced Leydig cell tumors in cryptorchid BALB/c mice was studied with the electron microscope. Changes in Leydig cell fine structure are apparent by 10 days after the s.c. implantation of a pellet of diethylstibestrol (DES). The smooth endoplasmic reticulum is diminished, and there is an increase in lipid droplets and free polysomes as compared with untreated cryptochid controls. These alterations persist as the Leydig cells proliferate to form focal areas of hyperplasia in the interstitial tissue. During this period of proliferation, activated macrophages containing large residual bodies appear among the Leydig cells. If DES treatment is continued for several months, malignant Leydig cell tumors, result. They are characterized by a nuclear and cytoplasmic pleomorphism of the Leydig cells and a decreased macrophage population. Virus-like particles are rarely seen within the cell during the period of tumorigenesis. Along with the reduction in smooth endoplasmic reticulum in the Leydig cells after DES treatment, evidence from the literature suggests that there is also a decrease in testosterone biosynthesis. However, it is not clear whether these two effect are correlated, since the level of the microsomal enzymes of steroid biosynthesis may vary independently of either the amount of smooth endoplasmic reticulum or the level of androgen secretion. The increase in lipid droplets seen in Leydig cells after DES treatment suggest the accumulation of precursors from the steroid biosynthetic pathway. The macrophages are though to represent scavenger cells, rather than a primary tumor cell population. The paucity of virus-like particles within altered Leydig cells implies that formed virus is not a prerequisite for tumorigenesis.

Animals↗