It is well known that prostaglandin F_2α (PG F_2α) regresses the corpora lutea in many animal species. However, the exact mechanism for the luteolysis of PG F_2α hasn't been well established yet. No reports so far have clarified the mechanism at all. Recently, we reported that the mechanism may be exhibited by the direct effect on the corpora lutea, based on in vivo experiments. The purpose of the present paper is to investigate the mechanism in mode detail in vitro on the above hypothesis.
Wistar strain immature rats, at 25 days of age, received 20 IU of pregnant mare serum. gonadotropin (PMS) subcutaneously, and 56 hours later, 40 IU human chorionic gonadotropin (hCG) intraperitoneously to induce superovulated ovaries. Six days after the hCG treatment, ovariectomy was carried out.
In a perifusion (continuous flow incubation) study, the TC-199 medium (1% glucose, pH 7.4) was continually saturated with a mixture of 95% oxygen and 5% carbon dioxide and pumped through polyethylene tubing to the incubation apparatus using a multichannel infusion pump. The incubation chamber, medium volume 0.5 ml, was set in a 37°C water bath. Hemiovaries were placed in an incubation chamber. After 20 minutes of preincubation, the experiment was performed. The perifusion was carried out with the medium for a control period of 60 minutes prior to the stimulation experiment, and then the media containing the stimulus were perifused for 4 hours or 1 hour. The flow rate of the medium was 10 ml/hour. The samples were collected every 20 minutes into a test tube, and the concentration of progesterone was measured. All values, therefore, represented progesterone secreted pg/ml/mg tissue/20 min. and indicated the mean of the duplicate study.
In the first long-term stimulation experiment, the perifusion was done with media containing a concentration of PG F_2α 20 μg/ml and hCG 1, 10,100 IU/ml for 4 hours. In the control group, the value of progesterone indicated a low level and showed no significant change. In the PG F_2α 20 μg/ml group, the value increased gradually according to stimulation, and then after 2.5 hours of stimulation it reached at a plateau, 3.8 times higher than that in the control period. In the hCG 1 IU/ml group, there were no apparent changes during stimulation. On the other hand, in the hCG 10 IU/ml group, the value increased continuously and significantly during stimulation. In the hCG 100 IU/ml group, in contrast with the hCG 10 IU/ml group, the value increased precipitously and arrived at a plateau after 1.5 hours of stimulation. Its value was 8.8 times higher than the value in the control period. In every group except for the control group, it was revealed that PG F_2α and hCG stimulated the corpora lutea to secrete progesterone.
In the next short-term stimulation experiment, perifusion was carried out with media containing the stimulus for one hour which was followed by media containing no stimulus for 3 hours. In the control group, the value showed a low level and indicated no remarkable change. In the hCG 10 IU/ml group, the value increased gradually according to stimulation. Furthermore, it increased continuously after the termination of stimulation and reached at a high level plateau, 5.1 times higher than that in the control period. In the PG F_2α 20 μg/ml group, 60 minutes after stimulation the value reached a peak 2.2 times higher than the control period value, and then in spite of the termination of stimulation, it remained at 1.7 times higher than the control period value for about 2 hours. In the PG F_2α 200 μg/ml group, the value reached a peak at 40 minutes after stimulation. Thereafter, it decreased suddenly after the termination of stimulation and remained at 1.6 times higher than the control period value.

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It is well known that the mediobasal preoptic-hypothalamic area plays an important role in the control of gonadotropin secretion from the anterior pituitary gland. Recently, many studies have demonstrated that catecholaminergic mechanisms are involved in the control of this process. However, the way in which the midbrain ascending pathways are linked with the forebrain structures which are critical for gonadotropin secretion is not yet clear. The purpose of the present study is to investigate the involvement of the midbrain ascending pathways in the control of preovulatory surges of gonadotropin and PRL.
Adult female rats of Wistar strain served as experimental animals. Each surgical operation was stereotaxically performed under ether anesthesia. Blood samples were collected without anesthesia through the indwelling atrial cannulae for the determination of serum LH, FSH and PRL by radioimmunoassay.
(1) The bilateral coronal transections, which were made in the midlateral hypothalamus at 1100-1330 h on proestrus, blocked ovulation in 6 out of 9 animals (p<0.05) and significantly suppressed the proestrous surges of serum LH, FSH and PRL when compared to the sham-transected animals. Similar coronal transections made in the anterolateral hypothalamus (ALH) blocked ovulation in all 6 animals (p<0.01) and eliminated the proestrous surges of serum LH, FSH and PRL. However, the bilateral coronal transections made in the lateral forebrain failed to block ovulation.
(2) The placement of ALH transections failed to block ovulation, which is induced by electrochemical stimulation of the diagonal band of Broca (DBB) in pentobarbital-blocked proestrous rats, without affecting the DBB stimulation-induced LH and FSH surges and PRL suppression.
(3) Following ALH transections, which were made at 1100-1330 h on proestrus, norepinephrine content in the preoptic-anterior hypothalamic area and dopamine content in the midposterior hypothalamus was decreased and increased, respectively, at 1700-1800 h on the same day.
(4) A single injection of norepinephrine bitartrate (40 μg) into the third ventricle of the ALH-transected proestrous rats induced ovulation in 5 out of 7 animals (p<0.05), of which 5 ovulating animals showed significant increases in serum LH, FSH and PRL values when compared to the animals injected with saline, while the intraventricular injection of dopamine hydrochloride (40 μg) induced ovulation in only 1 out of 6 animals.
(5) In the ovariectomized rats, the placement of ALH transections resulted in a significant increase in serum LH value (p<0.05), whereas it did not affect serum FSH and PRL values. The pulsatile secretion of serum LH was maintained after the ALH transections.
The present study suggests that the lateral hypothalamus mediates the ascending catecholaminergic inputs to the preoptic-hypothalamic area for inducing the proestrous surges of gonadotropin and PRL. The finding that the ALH transection-blocked ovulation recovered effectively by intraventricular NE may indicate the importance of the ascending noradrenergic system in the control of this process. On the other hand, serum LH values in the ovariectomized rats was significantly raised by ALH transections, which may suggest that the involvement of the midbrain ascending pathways in the control of gonadotropin and PRL is under the influence of ovarian steroids.

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The studies on the role of the midbrain ascending pathways in the proestrous surges of gonadotropin and PRL have recently appeared, suggesting the importance of monoamine pathways in this process. In the preceeding study, it was suggested that the ascending noradrenergic system plays a stimulatory role in the induction of preovulatory surges of gonadotropin and PRL. The purpose of the present study was to investigate the noradrenergic mechanisms in the lower brain stem which are involved in preovulatory surges of gonadotropin and PRL.
Adult female rats of Wistar strain served as experimental animals. Each surgical operation was stereotaxically performed under ether anesthesia. Blood samples were collected without anestheisa through the indwelling atraial cannulae for the determination of serum LH, FSH and PRL by radioimmunoassay.
(1) The bilateral electrolytic lesions, which were made in the ventrolateral part of the medulla oblongata at 1100-1330 h on proestrus, blocked ovulation in 5 out of 7 animals (p<0.05) and significantly suppressed the proestrous surges of serum LH, FSH and PRL when compared to the sham-lesioned animals, while the lesions made in the dorsomedial part of the medulla oblongata blocked ovulation in only 1 out of 5 animals.
(2) The bilateral implants of diethyldithiocarbamate (DDC), an inhibitor of norepinephrine synthesis, into the ventrolateral part of the medulla oblongata, which were made at 1100-1330 h on proestrus, blocked ovulation in 6 out of 9 animals (p<0.05) and significantly suppressed the proestrous surges of serum LH and FSH, while the DDC implants into the dorsomedial part of the medulla oblongata blocked ovulation in only 1 out of 5 animals.
(3) The unilateral implants of ovarian steroids (estradiol benzoate or progesterone) into the ventrolateral part of the medulla oblongata failed to increase or decrease serum LH, FSH and PRL values at least within 6 hours in the ovariectomized, estrogen-primed rats.
(4) The unilateral electrochemical stimulations of the ventrolateral part of the medulla oblongata failed to induce ovulation and the surges of serum LH and FSH in the pentobarbital-blocked proestrous rats, although the stimulations slightly suppressed the serum PRL value.
The present study suggests that the ventrolateral part of the medulla oblongata is involved in the induction of the proestrous surges of LH, FSH and PRL. The finding that the DDC implants into the ventrolateral part of the medulla oblongata significantly suppressed the proestrous surges of LH and FSH indicates that the noradrenergic system arising in the Al NE cell group may play an important role in the induction of the proestrous gonadotropin surge. However, the induction of the gonadotropin surge was not obtained from the implants of ovarian steroids into, nor the electrochemical stimulations of, the ventrolateral part of the medulla oblongata.

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Extracts of human term placenta were fractionated by Sephadex G-75 gel filtration and assayed for immunoreactive ACTH. Both high and low molecular weight protein fractions were detected to be immunologically reactive toward anti-human ACTH (1-39α) antibody. For the extraction of low molecular weight ACTH from human term placenta (pl. -ACTH), a glacial acetic acid-acetone mixture was employed, while a pH 3.0-HCl solution was used for high molecular weight immunoreactive ACTH.
The high molecular weight immunoreactive ACTH fraction (F-I), co-eluted with horse hemoglobin from a Sephadex G-75 column in 0.1M acetic acid, was essentially devoid of low molecular weight materials as revealed by polyacrylamide gel disc electrophoresis at pHs 9.5 and 4.3.
Tryptic digestion of F-1 at pH 8.1 and 37°C for 4 hr with E/S of 1/100, followed by fractionation with a Sephadex G-75, resulted in the formation of lower molecular weight fragments. One fragment was eluted at the same position as that of porcine ACTH with a recovery of 86% of immunoactivity of F-I. Another fragment which was eluted last exhibited positive β-endorphin receptor binding activity.
These results suggest the presence of a common precursor protein to ACTH and β-endorphin in human term placenta.

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Recently major resection of the pancreas has been employed for radical treatment of not only pancreatic carcinoma but also benign pancreas disease, such as chronic pancreatitis. Because it is reported that the amount of insulin required to control glycosuria after partial pancreatectomy is much greater than that needed after total pancreatectomy, it is believed that the pathophysiology of diabetes developed after partial pancreatectomy is considerably different from that following total pancreatectomy. However, metabolic changes have not been investigated in detail after partial pancreatectomy, although they have been widely investigated after total pancreatectomy.
In the present experiment, changes in glucose tolerance and the Langerhans islet were investigated after major resection of the pancreas in dogs to elucidate the pathophysiology of carbohydrate metabolism.
Immediately after a 90% or more pancreatectomy, diabetes developed, and the size and volume density of the islet was markedly decreased, accompanyied by severe degeneration of B, A and D cells, which was dominant seven weeks or more after the pancreatectomy.
During the early periods after 70-90% pancreatectomy, glucose tolerance was maintained within the normal range. Six to nineteen weeks later, so-called Sandmeyer's diabetes developed. Within five weeks after the diabetes developed, the size and volume density of the islet slightly increased. B cells in the remnant pancreas decreased in number and showed moderate degeneration, but both A and D cells were observed in the central part of the islet and increased in number. Five weeks or more after the diabetes developed, the size and volume density of the islet slightly decreased, with marked hydropic degeneration of B cells, similar to findings within seven weeks after 90% or more pancreatectomy.
After resection of less than 70% of the entire pancreas, diabetes did not develop, glucose tolerance was maintained well, and the islet was maintained at almost normal size and the volume density of the islet moderately increased, without any significant changes of B, A and D cells.

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In order to investigate the abnormalities of thyroid hormone secretion and metabolism in patients with chronic renal failure, plasma thyrotropin releasing hormone (TRH), thyrotropin (TSH), thyroxine (T4), 3, 3', 5-triiodothyronine (T3), 3, 3', 5'-triiodothyronine (rT3), 3, 3'-diiodothyronine (3, 3'-T2), free thyroxine (FT4) and thyroxine binding globulin (TBG) levels were measured by each radioimmunoassay and thyroxine binding prealbumin (TBPA) by the immunodiffusion method in 67 patients on hemodialysis. The effects of hemodialysis on thyroid hormone levels were also evaluated by measuring each parameter before and after hemodialysis in 12 patients. In 8 patients, thyroid hormone levels were serially measured for 16 weeks before hemodialysis. The following results were obtained.
1. Mean plasma TRH and TSH concentrations were 17.3 ± 4.0 pg/ml (Mean± SE) and 2.5±0.80μU/ml, respectively. These values were essentially the same as those in agematched control groups.
2. Mean plasma T4, FT4 and T3 concentrations were 5.9 ± 0.24μg/dl, 1.4 ± 0.08 ng/dl and 97 ± 5.0 ng/dl, respectively, and all these values were lower than normal. These differences were statistically significant (P<0.001).
3. Mean plasma rT3 concentration was 25.6± 1.2 ng/dl and 3, 3'-T2 was 8.1 ± 0.39 ng/dl, which was higher than normal (P<0.005).
4. Mean TBG concentration was 20.2 ± 0.67 μg/ml, whereas TBPA was 18.9 ± 1.6 mg/dl, lower than normal (P<0.001).
5. There were no significant sex differences between thyroid hormone levels. Mean plasma rT3 value increased with aging. Mean plasma T4 and T3 had a tendency to fall with duration of hemodialysis; rT3, however, tended to increase.
6. Neither thyroid hormone levels (T4, T3, rT3 and 3, 3'-T2) nor blood urea nitrogen and creatinine amounts correlated with each other. Minimal correlation was shown between rT3 or 3, 3'-T2 and T3, while there was an inverse correlation between rT3/T3 and T4 (P<0.01).
7. Hormone levels obtained before and after hemodialysis demonstrated that rT3, 3, 3'-T2 and FT4 varied after hemodialysis, but on long-term hemodialysis, thyroid hormone levels were stationary.
These results suggest the following :
The hypothalamic function in the cases of chronic renal failure seems to be normal, whereas the pituitary is quite variable, with some normal and some abnormal. Also, thyroid hormone secretion may be disturbed.
Thyroid hormone metabolic abnormalities may depend on metabolism in a number of peripheral organs, affecting either a deiodinated pathway from T4 to T3 or rT3. These metabolic abnormalities are modified by single hemodialysis and tend to be prevented during the course of long-term hemodialysis.

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The precipitating reaction between solubilized thyroid membrane fractions (microsomal and plasma membrane fractions) and sera from patients with autoimmune thyroid disease was studied by gel immunodiffusion.
Thyroid membrane antigens (100 mg wet weight) were solubilized by the addition of one ml of 0.5% Triton X-100 in PBS (pH 7.2).
Antigenic activity of the solubilized thyroid membrane antigens was confirmed by inhibition of the immunofluorescence antibody technique with an unfixed thyroid tissue section and microsomal hemagglutinin test (MCHA).
Nine out of 15 Hashimoto sera and 7 out of 15 Graves sera showed one or two precipitin lines which were different from those of the thyroglobulin - antithyroglobulin system.
All cases had positive MCHA at titres greater than 1 : 10,000. Precipitin lines of Hashimoto sera and Graves sera with solubilized thyroid membrane antigens fused in a complete immunological identity.
Precipitin lines of both microsomal and plasma membrane fractions with patients' sera also fused in a complete immunological identity.
Two cases showed two precipitin lines which were different from those of the thyroglobulin - antithyroglobulin system.
These two lines together fused with one precipitin line observed in another case.
The present results indicate that : 1) There is no difference in precipitating antibodies to solubilized thyroid membrane fractions between Hashimoto and Graves sera. 2) There is no difference in antigenicity between solubilized microsomal fractions and plasma membrane fractions. 3) Some cases had antibodies to an unknown subcomponent of the thyroid membrane fraction.

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Since human serum thyroglobulin (HSTG) could be measured by radioimmunoassay (RIA), it has been found to increase in various thyroid disorders providing a new aspect on thyroid pathophysiology. It is not easy to make a diagnosis of thyroid carcinoma, follicular adenoma, nontoxic multiple nodular goiter and cyst among thyroid diseases presenting thyroid nodules, prior to obtaining histological findings. The purpose of this study is to evaluate the diagnostic significance of HSTG in patients with these four diseases.
HSTG levels were measured by RIA in 27 patients with thyroid carcinoma, 12 with follicular adenoma, 8 with nontoxic multiple nodular goiter and 16 with cyst.
The HSTG of patients with thyroid carcinoma was 223±632 ng/ml (mean±SD), that in follicular adenoma 148±72 ng/ml, that in nontoxic multiple nodular goiter 103.8±127 ng/ml and that in cyst 114.3±201.9 ng/ml. The HSTG of all patients with follicular adenoma elevated above normal, and that in the other three diseases ranged from normal to very high levels. The HSTG level of six patients with non-metastatic, well-differentiated thyroid carcinoma was within the normal range, (32.4±15.4 ng/ml, mean±SD, n=7). On the other hand, the HSTG level of all patients with metastatic, well-differentiated thyroid carcinoma elevated above normal (312.2±758.8 ng/ml, mean±SD, n=18).
The size of the thyroid nodules in patients with non-metastatic, well-differentiated thyroid carcinoma was 5.0±5.0 cm² (mean±SD, n=7, maximum diameter X minimum diameter in cm) and that in metastatic carcinoma was 34.8±33.7 cm² (mean±SD, n=18). There was a significant relationship between HSTG and the size of the nodules in well-differentiated thyroid carcinoma (p<0.005).
Performing ^99mTc and ²⁰¹Tl scintigraphies for patients with these thyroid nodules, which discriminate nontoxic multiple nodular goiter and cyst from thyroid carcinoma and follicular adenoma, the above results may lead us to the following diagnostic evaluations of patients with thyroid nodules. The patients with a cold nodule in these scintigraphies are considered to have nontoxic multiple nodular goiter or cyst regardless of HSTG levels. On the other hand, the patients with a hot nodule in ²⁰¹Tl scintigraphies are considered to have non-metastatic well-differentiated carcinoma in cases of normal HSTG levels and to have metastatic well-differentiated carcinoma or follicular adenoma in cases of elevated HSTG levels.
A well-differentiated thyroid carcinoma was found to change to an anaplastic one in one patient, whose elevated HSTG level returned to normal concomitantly. Five patients with follicular adenoma were put on triiodothyronine therapy. In one of them, the size of the thyroid nodule decreased remarkably and the elevated HSTG level was restored to normal at the same time.
These results suggest that HSTG, as well as thyroid scintigraphies, is useful for the differential diagnosis of these thyroid nodules and helpful for understanding their pathophysiology.

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Recently, measurements of oxytocin content in serum have been reported from several institutes using radioimmunoassay (RIA) techniques. But these assay values showed significant differences, and the plasma levels of this hormone related to pregnancy and labor are still unclear. In order to clarify those problems, a sensitive and specific RIA system has been developed, and moreover, in this RIA system, any purification or extraction procedures of plasma samples were unnecessary.
Antiserum against oxytocin was produced in New Zealand white rabbits with multiple intradermal and intrasplenal injections of oxytocin-bovine serum albumin (BSA) complex with complete Freund's adjuvant. For the coupling of oxytocin with BSA, water soluble carbodiimide was used. After twelve weeks from the first immunization, antiserum capable of binding with 45-50% of¹²⁵ I-oxytocin at a final dilution of 1 : 72000 was obtained.
Iodination of synthetic oxytocin was done with the chloramine T method with some modifications, and the purification of iodinated oxytocin was achieved by using a Sephadex G-25 fine gel filtration (0.8 X 10 cm) eluted with 0.1M acetic acid. Further purification with a Dowex ion exchange column chromatography gave no benefit on this assay system.
In the RIA procedure, ¹²⁵I-oxytocin was added after a 24 hour-preincubation, and a double antibody method was used for the separation of B/F hormones. All measurements were done in duplicate, and 0.05M phosphate buffer pH 7.5 containing 0.01M EDTA-2Na, 0.015M NaN₃, 2.5 X 10^-5M o-phenanthlorine and 0.1% human serum albumin was used.
A linear dose response curve was obtained at an oxytocin concentration between 250 μU/ml and 16 μU/ml, and the minimal detectable dose was 2 μU/ml.
Cross-reactivities with Arg-vasopressin and Lys-vasopressin were less than 0.2%. The values of inter-assay coefficient of variation obtained from five different assays with two pooled plasma were 18.6% and 15.8%. The values of intra-assay coefficient of variation obtained from five different concentrations of pooled plasma were less than 4.9%.
The recovery rate was improved with the immobilization of plasma samples, and in this RIA system, anti-oxytocin serum, normal rabbit serum and anti-rabbit gamma globulin goat serum as well as plasma samples were used after immobilization.
The serial dilution of pregnant women's plasma gave reasonable assay values. From these results, this RIA system is thought to be satisfactory for measuring oxytocin in plasma.
Blood samples were obtained from normal pregnant women at Osaka University hospital at 2-3 pm. Sampling was done from the antecubital vein with a previously cooled and heparinized syringe, and the blood was immediately mixed with an ice cold solution, containing 0.01M EDTA and 2.5 X 10^-5M o-phenanthlorine for the inhibition of oxytocinase activity. After immobilization, the plasma samples were kept in a deep freezer (-20°C) and in this condition no change was observed in the assay values at least within one month.
The plasma oxytocin levels during normal pregnancy, evaluated by this RIA system, showed a gradual rise towards the estimated delivery date, and the mean value at the 10th month of pregnancy was 12.5 μU/ml.

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The method of determining catecholamines has been studied, and such sensitive methods as radioenzymatic assay and gas chromatography are used for their measurement, but these techniques are not widespread because of their complexity.
In this study, we developed a high pressure liquid chromatographic method with a fluorescent reaction system for the determination of plasma, urine and tissue catecholamine concentrations.
The eluates from plasma, urine and tissue were chromatographied on a Hitachi #3011C Gel (2.6 X 250mm) and reacted to the mixed solution of 0.1M KH₂PO₄, 0.2M K₂HPO₄ and 0.0075% K₃Fe (CN) ₆ (the first solution), followed by the mixture of 0.1% ascorbic acid and 5N NaOH. A stock solution (0.1mg/ml of norepinephrine, 0.lmg/ml of epinephrine and 2.0mg/ml of dopamine) was stored in 0.2N CH₃COOH at 4°C, and was diluted at 1/5,000-1/40,000 when needed.
Recovery of norepinephrine after extraction was 54.3-58.9% and that of epinephrine was 49.2-55.8%. The accuracy was acceptable and sensitivity was 30-50pg. The mean plasma norepinephrine concentrations (p-NE) were 105.6 ±6.8pg/ml in normal subjects and 157.7 ± 74.5pg/ml in essential hypertensives (EH). The mean epinephrine concentrations (p-E) were 30.8 ± 16.3pg/ml in normals and 20.0 ± 11.9pg/ml in EH. PNE increased after standing or after furosemide plus 2-hour standing, but p-E did not increase as much as p-NE. Catecholamine concentration in the rat brain and adrenal medullary tissue could be determined by the use of this method.

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