Oddvar Naess

Androgen receptors in the rat adrenal gland.

Abstract Cytosol fractions from rat adrenal glands contain specific androgen binding proteins showing high affinity ( K D ~ 3.9 × 10 9 M) and limited capacity (22 fmol/mg protein) for testosterone and with physicochemical properties identical to those of androgen receptors in the prostate, epididymis and testis. The androgen receptor complexes have a sedimentation coefficient of 7–8 S by sucrose density gradient centrifugation, and a mobility relative ( R F ) to bromophenol blue of 0.5 by electrophoresis in 3.25% polyacrylamide gels containing 0.5% agarose. Isoelectric focusing in agarose-polyacrylamide gels indicated a pI of 5.9. Androgen binding to these receptors is destroyed by heating at 45°C for 30min. by incubation with p -chloromercuriphenyl sulphonate (2 mM) or protease, but not by RNase. Testosterone (T) and 5α-dihydrotestosterone (DHT) were the most potent androgens in displacing [ 3 H]-T binding. However, 4-androsten-3,17-dione (A) and 5α-androstan-3α,17β-diol (Adiol) also significantly displaced [ 3 H]-T binding from the receptor. The reasons for this are the high activities of 17β-OH steroid dehydrogenase (converting A into T) and 3α-oxido-reductase (converting Adiol into DHT) in the cytosol fractions. These enzymes are active even at 0°C. Estradiol-17β in high concentrations (100-fold excess) also reduced the [ 3 H]-T binding, by approximately 60%, whereas corticosterone was without effect. The androgen receptors were found in similar concentrations in the adrenal cytosol of male and female rats, but were virtually absent in cytosol fractions obtained from rats with testicular feminization syndrome ( tfm rats). The presence of specific androgen receptors in the adrenal gland of normal rats indicates that androgens may affect adrenal steroid production by a direct action.

Receptors for 17β-estradiol in prolactin-secreting rat pituitary cells

Abstract Estrogens stimulate prolactin (PRL) synthesis by GH3 cells, a clonal strain of rat pituitary cells grown in culture. At 4°C the binding of [3H] 17β-estradiol to monolayer cultures of GH3 cells was specific and of limited capacity, with half-maximal and maximal binding after 1–2 h and 12 h, respectively. Scatchard analysis showed one single class of binding sites with Kd = 3.1 × 10−10 M and n = 309 × 10−15 mol 17β-estradiol/mg cell protein, calculated to give approx. 25,000 binding sites per cell. At 4°C We suggest that the action of 17β-estradiol on GH3 cells involves an initial binding of the steroid to specific receptors in the cytoplasm, followed by transport of a fraction of the hormone—receptor complexes to the nucleus involving a temperature-sensitive step.

Endocrine status of the testicular feminized male (TFM) rat

Abstract The plasma levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T), androstenedione (A), dihydrotestosterone (DHT), corticosterone and corticosterone binding globulin (CBG) were determined in 160- and 350-day testicular feminized male (tfm) and normal littermate (NL) rats. In the younger group the concentrations of T, A, DHT and 5α-androstan-3α,17β-diol (Adiol) were also determined in testis tissue. Tfm rats showed a greatly elevated plasma LH indicating lack of androgen feedback. Plasma FSH, however, was normal in both age groups, suggesting that inhibin production from tfm testes was relatively unaffected. Intratesticular concentrations of A were greatly elevated in the tfm animals at both ages whilst the testicular levels of T were highly reduced when compared to NL rats. This indicates a gonadal deficiency of the 17β-hydroxy steroid dehydrogenase (17β-HSD). Furthermore, the relatively low T: DHT ratio and high concentrations of Adiol in the tfm testes confirmed previous reports that the high 5α-reductase typical for the immature rat testis is maintained into adulthood in rats with the tfm condition. A considerable degree of peripheral conversion (A → T) probably helps to maintain normal or supranormal plasma levels of T. Gonadectomy rendered all plasma androgens undetectable, indicating that the adrenal contribution was negligible. Increasing age (350 days) was associated with a marked increase in circulating androgens in tfm rats. This is probably the reason for the observed significant reduction in circulating LH in this age group when compared to the 160-day animals. The aging tfm rat is predisposed to testicular tumors which, on the basis of histology, specific [125I]hLH binding and in vitro responsiveness to hCG, appear to be of Leydig cell origin. Microflow fluorometry (MFF) of tfm testis cell suspensions revealed the presence of haploid cells, suggesting that meiosis proceeds to a limited extent. Plasma corticosterone levels in the tfm rat were normal although plasma CBG levels were highly significantly elevated at both ages when compared to the levels in NL rats. We suggest that the adrenal hyperplasia observed in tfm rats is secondary to reduced corticosterone production and diminished free corticosterone in the circulation.

Alterations in mucosal morphology and permeability, but no bacterial or endotoxin translocation takes place after intestinal ischemia and early reperfusion in pigs.

Ischemia and reperfusion of the gut may be an important etiological factor in the development of multiple organ failure. We have used a hemorrhagic and a superior mesenteric artery (SMA) occlusion shock model in pigs to estimate the effect of ischemia and reperfusion on intestinal morphology, mucosal permeability, and the occurrence of bacterial or endotoxin translocation. Mucosal ulceration and necrosis were found in the SMA shock model, while the morphological changes were less pronounced in the hemorrhagic shock model. Scanning electron microscopy showed shrinkage of the villi and plugging of the colonic crypts in both shock models. Enterocyte cell kinetics was investigated using 5-bromo-2′-deoksyuridine (BrdU) incorporation and immunovisualization by anti-BrdU antibodies. Cell renewal was almost completely lost from the jejunum to the rectum in both shock models. Intramucosal pH was measured using a tonometer placed in the terminal ileum. Segments of intestinal mucosa were mounted in Ussing chambers, and permeability was measured using radioloabeled probe molecules of differing molecular weights. Augmented molecular flux of inulin (Mr 5.000) and mannitol (Mr 182) and loss of short circuit current (Isc) and transepithelial potential difference (PD) were found in mucosae from both shock models. Endotoxin was demonstrated in the ascitic fluid in both shock models; 9.5 (2.7–14.3) (median and 95% confidence interval) EU/mL in the SMA occlusion model and 16.0 (4.9–29.4) EU/mL in the hemorrhagic shock model), but the levels were not significantly higher than in the control model 6.5 (4.3–34.0) EU/mL. In agreement with this, no pathogenic bacteria or endotoxin could be detected in the systemic circulation, portal blood, lymph, or in homogenates of mesenterial lymph nodes, liver, or lung. These findings suggest that there is no translocation of bacteria and endotoxin to the systemic circulation during intestinal ischemia and early reperfusion even though the mucosa morphology is highly aberrated and its permeability increased.

Ploidy Analysis by In Situ Hybridization of Interphase Cell Nuclei in Fine-Needle Aspirates From Breast Carcinomas: Correlation With Cytologic Grading

Fine‐needle aspirates from 54 breast cancer patients were investigated for numeric aberrations in chromosomes 6, 7, 12, and 17 by in situ hybridization (ISH) of interphase cell nuclei. Ploidy findings were compared with cytologic grading of tumors. Aneuploidy was found in 73% of cases. Chromosomes 6 and 7 showed numeric abnormalities in 63% and 62% of cases, respectively, whereas chromosome 17 retained a disome pattern in 2/3 of the tumors. Thirteen cancers (28% of 47 with four analyzed probes) had a normal signal number in all four chromosomes. In 17 (36%), all four had signal gain. Another 17 showed a mixed disome/aneusome pattern. They presented a continuum of increasing numeric abnormalities, 82% disomy for chromosome 17, and 13 of them were grade 2, indicating intermediate biologic properties. Correlation between grading and ploidy was good, with 10 of 11 grade 1 carcinomas showing diploidy, whereas 33 of 36 grade 2 and 3 tumors had numeric aberrations. Diagn. Cytopathol. 1997; 17:267–271.

nm23 protein expression in fine‐needle aspirates from breast carcinoma

nm23 has been recognized as a potential suppressor gene of metastasis. Reduced nm23 expression in breast carcinoma has been found to correlate with axillary lymph node metastases, high grade tumors, and shorter survival.

Assessing Invasion Criteria in Fine Needle Aspirates from Breast Carcinoma Diagnosed as DCIS or Invasive Carcinoma

OBJECTIVE To evaluate invasion criteria in fine needle aspiration cytology (FNAC) of histologically diagnosed breast ductal carcinoma in situ (DCIS) and invasive carcinoma and to evaluate their usefulness in identifying an invasive component in addition to DCIS. STUDY DESIGN The material consisted of 331 smears diagnosed as suspicious for or consistent with DCIS and in which histology had shown either DCIS or invasive ductal carcinoma. All smears were reevaluated for the following invasion criteria: invasion of fat or fibrous tissue fragments, fibroblast proliferation, cell-poor elastoid tissue fragments, tubular structures and intracytoplasmic vacuoles. RESULTS All invasion criteria except cytoplasmic vacuoles correlated with invasiveness, but none of them were found exclusively in invasive lesions. Pseudoinvasion in fibrous or fatty tissue fragments were found in 8 cases of histologic pure DCIS. One DCIS (0.4%) revealed > or = 2 invasion features as well as 22 invasive carcinomas (20.7%), representing 7.4% of all cases. CONCLUSION Using established invasion criteria, practically no pure DCIS lesion will be diagnosed as invasive on FNAC, but one will identify only a subset of cases harboring an invasive component.

Numerical aberrations of chromosome 17 in interphase cell nuclei of breast carcinoma cells: lack of correlation with abnormal expression of p53, neu and nm23 protein

The genes for p53, neu (c‐erbB‐2) and nm23 are all located on chromosome 17. Abnormal expression of their protein products is an important prognostic parameter. The aim of this study was to investigate if numerical aberrations of chromosome 17 are reflected in the expression of these markers. The immunohistochemical expression was analysed on histological specimens from 33 breast carcinomas. In situ hybridization (ISH) was performed on interphase cell nuclei in air‐dried fine‐needle aspirates from the same cases using a digoxigenin‐labelled α‐satellite probe for chromosome 17. ISH for chromosome 6, 7 and 12 was used additionally to give an estimate of ploidy. Of the carcinomas 76% were aneuploid, and numerical abnormalities of chromosome 17 were found in 34%. Abnormal p53 protein was expressed in 15% (five cases). All of these were aneuploid, but only one of them revealed aneusomy of chromosome 17. Neu overexpression was found in 18% of the tumours (six cases). Five of these were aneuploid, whereas two were aneusome for chromosome 17. Four cancers showed full (normal) expression of nm23 protein, whereas 29 had reduced expression. Reduced expression was found in 23 of 25 aneuploid tumours. Numerical aberrations of chromosome 17 were found equally in carcinomas with reduced and full nm23 protein expression. Abnormal numbers of chromosome 17 seem only to have a minor impact on these markers and are not reflected significantly in their expression.

Further characterization of the androgen receptor in rat testis

Immature rat testes contain a specific binding protein for testosterone (T) and 5alpha-dihyrotestosterone (DHT) with physico-chemical properties similar to the cytoplasmic androgen receptors in the epididymis and ventral prostate but different from the testicular androgen-binding protein (ABP). Like the androgen receptors in the prostate and epididymis, it has a sedimentation coefficient of about 7 S at low ionic strength, is eluted in or close to the void volume on Sephadex G-200 gel filtration (Stokes radius greater than 80 A), has an isoelectric point of about 5.6-6.0 (mean) 5.8 and a relative mobility (Rf) of 0.4 in 3.25% acrylamide gels. Following the injection of 3H-labeled testosterone, T and DHT are bound selectively by the receptor. Relatively more [3H]T than [3H]DHT is present in bound and free fractions as well as in total testicular 105,000 g supernatant. Similar results are obtained from testicular incubations with equimolar amounts of [3H]T and [3H]DHT at 0 degrees C in vitro. Saturation of receptor sites is achieved by incubation of testis supernatants with increasing amounts of [3H]T at 0 degrees C. The number of available binding sites following post-hypophysectomy regression is estimated to be about 9 fmoles/mg protein, and the apparent equilibrium constant of dissociation is 7 X 10(-10) M. The temperature stability and sulfhydryl dependence of the testicular androgen receptor are similar to androgen receptors in other organs. Binding is destroyed by heating the supernatants at 50 degrees C for 30 min and by exposure to p-chloromercuriphenylsulfonate (1 mM) at 0 degrees C for 60 min. Furthermore, like other androgen receptors, the half-time of dissociation of testicular androgen-receptor complexes at 0 degrees C is extremely slow (t1/2 greater than 35 h). Separation of seminiferous tubules from interstitial tissue showed that a major portion of these receptors were localized within the seminiferous tubules.

Cytologic features of ductal carcinoma in situ in fine‐needle aspiration of the breast mirror the histopathologic growth pattern heterogeneity and grading

Approximately 20% of the breast carcinoma cases detected on mammography screening represent ductal carcinoma in situ (DCIS). Cytopathologists are exposed to cytologic material from DCIS when nonpalpable, mammographic lesions are aspirated during the workup of organized and opportunistic mammography screening.