It is very important to detect the intracellular distribution and dynamics of estrogen receptor (ER) in order to make the mechanisms of estrogen action clear. In a recent report, monoclonal antibodies localized ER only in the nucleus of target cells under both the presence and absence of estrogen.
We purified cow and human uterine ER and induced specific polyclonal anti-ER anti-bodies. In this report, the specificity of immunological reactivity of anti-ER antibodies was examined, and the distribution of ER in the target tissues was detected by the indirect immunoperoxidase method.
The specific staining was observed in the nucleus and cytoplasm of the endo- and myometrium of cow, rat and human uterus. In the nontarget organs, such as kidney and muscle, the specific staining was not detected. The cytoplasm of MCF-7 cells and immature rat uterine tissues were mainly stained without estrogen; however, the nuclear staining in-creased obviously after the estrogen priming. The cytoplasmic and/or nuclear staining was positive in 63% of 21 human breast cancer cases. This immunohistochemical assay (IHC) method of ER measurement was compared with the DCC assay method, a biochemical assay based on hormone binding ability. The concomitant positivity or negativity in DCC and IHC were observed as 61.9% (13 cases) or 19.0% (4 cases), respectively. Therefore, these two methods showed the same assay results in 81.0% of human breast cancer tissues.

View full abstract

An enzyme immunoassay for serum 18-hydroxycorticosterone was established using alkaline phosphatase as a label. The antiserum for 18-hydroxycorticosterone was produced by immunization of rabbits with 18-hydroxycorticosterone 3- (o-carboxymethyl) oxime conjugated to bovine serum albumin. Sephadex LH-20 column chromatography was used to separate 18-hydroxycorticosterone from other steroids in serum samples. The minimal detectable amount of 18-hydroxycorticosterone was 50 pg/tube, and the measurable range was from 5 to 1000 ng/dl when a 1.0 ml serum sample was used. Intra- and inter-assay coefficients of variance were 5.0% (n=6) and 5.8% (n=6), respectively. In normal controls, the serum 18-hydroxycorticosterone level was 4.8 - 34.0 ng/dl (mean ± S.D. = 17.1 ± 9.0 ng/dl) on an unrestricted diet. Seven out of 8 patients with aldosterone-producing adenoma had above-normal serum 18-hydroxycorticosterone levels.
Serum 18-hydroxycorticosterone increased and decreased significantly following ACTH and dexamethasone administration, respectively. In essential hypertensive patients, serum 18-hydroxycorticosterone was high during a low-sodium diet and was suppressed remarkably by captopril. These observations support the previous reports that adrenal 18- hydroxycorticosterone synthesis is dependent on both ACTH and the renin-angiotensin system. The present method is sufficiently sensitive and producible, avoids the use of radioisotopes and is quite satisfactory for clinical use.

View full abstract

In this study, we established a method for the quantitative measurement of native adrenal steroids with GC-MS equipped with capillary column (cross-linked methyl silicone 25 m × 0.2 mm I.D., 0.11 m thin film). 1 ml of serum sample containing 5α-cholestane as internal standard (IS) was elicited by organic solvent using extrelunt column^R. These samples were derived by n-butylboronic acid, o-methylhydroxylamine and trimethyl-silylating agents, then were finally applied to GC-MS. The intensities of molecular ions were used for the measurement of the serum concentration of steroids. The molecular ion peaks of steroids were obtained at m/z460 (17α -hydroxyprogesterone; 17OHP), m/z548 (corticosterone; B), m/z470 (11-deoxycortisol; S), m/z417 (pregnenolone; PL), m/z372 (progesterone; PT), m/z558 (cortisol; F), m/z389 (dehydroepiandrosterone; DHEA), m/z371 (estrone; E1), m/z416 (estradiol; E2), m/z504 (estriol; E3), m/z389 (testosterone; T), m/z344 (androstenedione; A) and m/z372 (IS). The curve of calibration for each steroid showed good linearity. The sensitivities of the GC/MS method were less than 5 pg/one shot of each sample. The coefficients of variations of accuracies and precisions in this GC/MS method were less than 15% of each steroid. The samples from normal subjects after metyrapone and ACTH loading tests, and the patients of congenital adrenal hyperplasia showed a good correlationship between the data of GC/MS and the data of RIA after sephadex LH-20 columnchromatography. These results implied the usefulness of our system in clinical application. Moreover, this assay takes only 3 hrs. Thus it saves much time in comparison with the time-consuming radioimmunoassay system.

View full abstract

The immunological properties of thyroglobulins (Tg) of individual patients, obtained from a thyroid tumor and its adjacent tissue were compared, using conventional or monoclonal antibodies against human Tg.
The thyroid tumors studied were non-functioning thyroid carcinomas and functioning thyroid adenomas. In contrast to non-functioning tumors, Tg from the functioning tumors was generally iodinated at a level close to that of normal tissue, and Tg from the tissue adjacent to the tumors had a very low iodine content. The conventional antiserum and monoclonal antibodies, B2F, seemed to recognize the conformation of Tg, while C6G showed a high affinity to Tg even when unfolded or denatured.
In most cases, Tg isolated from the tissue adjacent to a tumor showed a higher affinity to antibodies than Tgs of the tumor tissue, as determined by the inhibitional effect of these Tgs against the binding of standard Tg and antibody. Furthermore, the Tg of the adjacent tissue was immunologically different in nature from the standard Tg obtained from a normal thyroid gland.
From these results, Tgs of tumor and the adjacent tissue in individual patients were heterogeneous in immunological property, regardless of iodine content.

View full abstract

A rapid, simple and sensitive immunochemical assay of pregnanediol 3-glucuronide (Pd-3G) in urine was established. The standard error range of the recovery test was within 0.8 μg/ml, and the coefficient variation of the within run and day to day precision tests were within 4%. Cross reactivity of various steroids exhibited less than 4%. A significant rise of Pd-3G (mg) /creatinine (g) coincided with the LH surge and continued during the luteal phase in the ovulatory menstrual cycle. In an anovulatory cycle, Pd-3G was found to be under 4 mg/g. This Pd-3G direct assay method could be widely used for assessing ovulation and luteal function.

View full abstract

To elucidate if the thyroid hormone acts directly on the ovary, the biocellular effect of L-thyroxine (T₄) on porcine granulosa cells cultured in vitro was investigated. Monolayer cultures of porcine granulosa cells obtained from small (1-2 mm), medium (3-5 mm) or large (6-11 mm) follicles were carried out in the presence of porcine FSH (100 ng/ml).
Concomitant treatment with T₄ promoted FSH-dependent morphological luteinization, i.e. alteration of immature granulosa cells obtained from small follicles to epithelioid form. T₄ also increased FSH-stimulated induction of hCG/LH receptor on immature granulosa cells. Furthermore, T₄ augmented FSH-mediated production of progesterone and estradiol by immature granulosa cells cultured in vitro. The concentration of T₄ to produce the maximal stimulatory effect was 10^-7M, demonstrating that optimal concentration of thyroid hormone is required for the expression of this stimulatory action.
Since T₄ alone demonstrated no effect on the differentiation of porcine granulosa cells and all the stimulatory effects of T₄ on the cells were observed with the concomitant treatment with FSH, this stimulatory effect of T₄ seems to have a permissive action on FSH-induced granulosa cell luteinization.
Although insulin showed a similar effect on porcine granulosa cells, no stimulation of estradiol production by porcine granulosa cells was observed with insulin in the culture system used in this study.
These results suggest that the thyroid hormone acts directly on the ovary and plays an important role in modifying the FSH-dependent cellular differentiation of immature granulosa cells.

View full abstract

The insulin-like activity of oxytocin in stimulating glucose oxidation in rat adipocytes has been demonstrated repeatedly in the last 20 years. Oxytocin binds to a specific cell surface receptor of adipocytes; however, little attention has yet been paid to the effect of oxytocin on glucose oxidation in other tissues. We have initiated studies into the metabolic-regulatory activity of oxytocin in insulin sensitive tissues and uterus.

View full abstract

We investigated the G₁ and S phases of synchronization using sodium n-butyrate, MTX and excess thymidine. The flow cytometry system was employed for cell cycle analysis while the receptor assay was adopted dextran coated charcoal (DCC) and the wash method.
The results were as follows :
(1) S phase synchronization by MTX was 138% (control 100%) and by excess thymidine in the block and release method 210%. G₁ phase synchronization by sodium n-butyrate was 140%.
(2) The progesterone receptor level, by E₂ priming increased to 1.45 fmol/ug DNA being more than five times that of the control PR.
(3) The estrogen receptor level increased to 18.29 fmol/ug DNA in the G₁ phase synchronization, seven times that of the control ER.
From this study, the functional increase of the steroid receptor was most significant in the G₁ phase.

View full abstract