The ultrastructural changes of ovariectomized rat endometrium were studied after the intraluminal administration of uterine high molecular RNA, for the purpose of understanding the action mechanism of uterine-RNA from the morphological standpoint. The following pertinent findings were obtained.
Increase in the height and the number of microvilli was observed in the luminal epithelial cells 12 hours after the injection of RNA.
There was also slight enlargement of cytoplasm which contained abundant free ribosomes with some polyribosomes, the granular endoplasmic reticulum with moderately dilated cisternae and a developed Golgi complex. Furthermore, the number of the mitochondria was also increased and the nucleoli were well defined.
On the contrary, the number of lipid droplets decreased slightly but the obvious interdigitation between the basal part of the luminal epithelial cells was noted.
After 24 hours, the above-mentioned changes in the organelles were more prominent in the luminal epithelial cells. The apical vesicles were recognized and large polymorphic structures, suggesting lysosome were also observed at this period.
These findings were similar to those observed in the endometrium after the administration of estradiol-17β.
The possible action mechanism of this uterine-RNA was discussed.

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The influence starvation and alloxan treatment on the butyrate metabolism, and the relationship between the butyrate metabolism and glycolysis were changed by the starvation and alloxan treatment in sheep liver slices have already been reported. Further study was designed to elucidate the relationship between the butyrate metabolism and TCA cycle, and how this relationship was influenced by the starvation and alloxan treatment in sheep liver slices.
The results obtained are summeri'ed as follows.
1) In liver slices of normally fed sheep, the ¹⁴C transfer from ¹⁴C-butyrate into glucose and NEFA was decreased and the formation of ¹⁴C-cholesterol, -triglyceride and -phospholipid from ¹⁴C-butyrate was increased by the addition of citrate or succinate. And the formation of ¹⁴CO₂ and ¹⁴C-phospholipid from ¹⁴C-citrate was increased and the ¹⁴C transfer from ¹⁴C-citrate into glucose, ketone bodies, cholesterol and triglyceride was decreased by the butyrate addition, and the formation of ¹⁴CO₂ and ¹⁴C-ketone bodies form ¹⁴C-succinate was increased and the ¹⁴C transfer from ¹⁴C-succinate into glucose, cholesterol, triglyceride and phospholipid was decreased by the butyrate addition.
2) In liver slices of starved sheep, the ¹⁴C tranfer from ¹⁴C-butyrate into CO₂, cholesterol, triglyceride and phospholipid was increased and the formation of ¹⁴C-glucose and -NEFA was decreased by the addition of citrate or succinate. And the formation of ¹⁴CO₂, ¹⁴C-glucose and -phospholipid from ¹⁴C-citrate was increased and the ¹⁴C transfer from ¹⁴C-citrate into ketone bodies, cholesterol and triglyceride was decreased by the butyrate addition, and the formation of ¹⁴CO₂, ¹⁴C-glucose and -ketone bodies form ¹⁴C-succinate was increased and the ¹⁴C transfer from ¹⁴C-succinate into cholesterol, triglyceride and phospho-lipid was decreased by the butyrate addition.
3) In liver slices of alloxan diabetic sheep, there were no effects of the citrate addition on the ¹⁴C transfer from ¹⁴C-butyrate into CO₂, ketone bodies and lipid fractions and also there were no effects of the butyrate addition on the ¹⁴C transfer from ¹⁴C-citrate into all these fractions. But the formation of ¹⁴C-glucose and -NEFA from ¹⁴C-bytyrate was decreased and the ¹⁴C transfer from ¹⁴C-bytyrate into cholesterol, triglyceride and phospholipid was increased by the succinate addition. And the formation of ¹⁴CO₂ and -ketone bodies from ¹⁴C-succinate was increased and the ¹⁴C transfer from ¹⁴C-succinate into glucose, cholesterol, triglyceride and phospholipid was decreased by the butyrate addition.
4) In liver slices of normally fed, starved and alloxan diabetic sheep, there were no effects of the addition of citrate or succinate on the formation of ¹⁴C-ketone bodies from ¹⁴C-butyrate, and also there were no effects of the butyrate addition on the ¹⁴C-NEFA formation from ¹⁴C-citrate or -succinate.
As mentioned above, it may be deduced that there are some relationships between the butyrate metabolism and TCA cycle and these relationships between the butyrate metabolism and TCA cycle were changed by the starvtion or alloxan treatment in sheep liver slices.

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Goitrous patients (37 with chronic thyroiditis, 38 with Graves' disease, 3 with subacute thyroiditis, 8 with simple diffuse goiter and so on) were injected intrathyroidally with 40 mg of triamcinolone acetonide (Kenacort-A) or methyl-prednisolone (Depot-medrol) in 3 to 30 or more doses at intervals of 2 to 4 weeks.
Except for simple diffuse goiter, marked reduction of goiter (average 40-35% of the initial size) was observed from 1 to 2 months and continued during the treatment. Symptoms caused by thyromegaly disappeared promptly although hardness of the goiters increased. Prominent histological changes in biopsy specimens were diminution of cell infiltration and restoration of follicular (cell) structure.
After cessation of the treatment, the reduction of goiter size persisted for 6 months and up to at least 2 years in 17 (58%) of 29 cases with chronic thyroiditis and 7 (37%) of 19 cases with Graves' disease. The patients with remission seemed to have smaller goiters initially, greater reduction in size during treatment, and those with Graves' disease were older than those with relapse. Serum level of free thyroxine index, autoantibody titer to thyroglobulin and thyroid microsomal fraction, and of gammaglobulin, as well as erythrocyte sedimentation rate showed slight and insignificant changes before and after the treatment, or between groups with and without relapse.
It is concluded that intrathyroidal administration of long-acting glucocorticoid can reduce thyromegaly and the resulting symptoms in chronic thyroiditis, subacute thyroiditis and Graves' disease promptly and sufficiently without side effects even after prolonged administration, but that such a therapy does not seem to alter the potenciality of the disorders, especially of Graves' disease.

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Radioimmunoassay for estrone (E₁), estradiol-17β (E₂) and estriol (E₃) in human urine and amniotic fluid is described. A sample volume of 0.002-4.0 ml was used for the assay. As indicators, each tritiated estrogen of glucuronides, dominant conjugates in urine and amniotic fluid, was added to the samples to correct for procedural losses. Conjugated estrogens of glucuronide (G) and sulfate (S) type were hydrolyzed with 15 vol% HCl at 100°C for 60 minutes, and resulting free estrogens were extracted with ether. The dried extracts were applied to microcolumns of Sephadex LH-20, then E₁, E₂ and E₃ were separated by eluting with benzene-methanol (85 : 15) mixture. A 1/2 aliquot of the eluate was used for measuring recovery of the added tracer, and the remainder after being added with tritiated free estrogen was dried and incubated with the antiserum in a dilution of the borate buffer (pH 8.0) containing 0.06% bovine serum albumin (BSA) and 0.05% bovine gamma globulin. The free and bound steroids were separated by 50% (NH₄) ₂SO₄, and tritium activities in the supernatant were counted. The antisera used for the assay were anti-E₁-17-BSA for E₁, anti-E₂-6-BSA for E₂ and anti-E₃-6-BSA for E₃, which had been prepared by us. In urine, all recoveries were 65.0±5.6 (mean± S. D.) % for E₁-3-S, 68.8±7.2% for E₁-3-G, 64.5±5.8% for E₂-17β-G and 41.1±9.1% for E₃-16-G. In amniotic fluid, the recoveries were almost equal to those of the urine. Water blank in each estrogen assay was about 4 pg/sample. Accuracy and precision of the method were satisfactory. The results determined by this radioimmunoassay were in good correlation with those determined by Brown's colorimetry at high estrogen concentration in urine. As this method is accurate and highly sensitive, we can determine estrogen values of low level or with small assay volume in both urine and amniotic fluid.

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Double antibody radioimmunoassay (RIA) for human urinary follicle stimulating hormone (HUFSH) was studied. RIA kit for urinary FSH was purchased from Istituto Farmacologico Serono, Rome, Italy. The assay was carried out with the pre-precipitation technique described by Hales and Randle for Insulin. Purified urinary FSH, 920 IU (immunopotency) /mg, contaminated with less than 11.2 IU (immunopotency) of LH/mg, was labeled with ¹²⁵I to specific activity of 100-120 μCi/μg. The 2nd IRP-HMG kindly supplied by World Health Organization, the Medical Research Council, London, was employed as the standard. The elution patterns of FSH-¹²⁵I from DEAE-C columns are shown in Fig. 1. Fractions, eluted with sodium potassium phosphate buffers containing 0.01M and 0.05M NaCl, were used in the radioimmunoassay. Antiserum to urinary FSH was used in a final dilution of 1 ampoule/120ml. Equal volumes of antiserum to urinary FSH and precipitating antiserum to rabbit gamma globulin were pre-incubated together for 2 days at 4°C. Aliquots of 0.2ml of the pre-incubated antisera were added to the tubes containing 0.1 ml (approximately 10,000 cpm) of labeled hormone, 0.1 ml of serum from a normal rabbit (NRS) in a dilution of 1 : 80 and 0.2 ml of urinary sample or standard. The mixtures were incubated for 4 additional days. Highly purified human chorionic gonadotropin (12,000 IU/mg) and human pituitary luteinizing hormone (LER 960) did not significantly inhibit the binding between anti-FSH serum and ¹²⁵I-FSH, even at concentrations of 1,000 IU/ml, and 500 ng/ml, respectively (Fig. 2). The effects of dialysis of unprocessed urine and urinary extract (fraction A) on this RIA system for FSH are shown in Figs. 4 and 5. The dilution curve of unprocessed urine was affected by dialysis, but that of urinary extract was not (Fig. 5). Fig. 6 shows that the inhibition slope resulting from fraction A was parallel only with that of the standard hormone, 2nd IRP-HMG. Although the inhibition curve by NaCl could be drawn with this RIA system, it was not parallel with that of 2nd IRP-HMG and the inhibitory effect of NaCl was lost after dialysis (Fig. 7). Dialysis did not change the dose response curve of 2nd IRP-HMG. The dilution curve of ethanol precipitate was parallel with that of 2nd IRP-HMG, but the binding capacity of ethanol precipitate was slightly larger than that of urine which was not treated with ethanol (Fig. 8). The accuracy of measurement was determined by adding 2nd IRP-HMG to dialyzed urine. Mean recovery was 98.9% (Fig. 9). To evaluate reproducibility, the same urine samples were analyzed in 3 replicate assays. Means, standard deviations and coefficients of variation are shown in Table 1. The mean value of the coefficients of variation was 15.5%. A comparison of UFSH-RIA and bioassay (Igarashi-McCann assay) potencies of urine (fraction A) was carried out. Correlation coefficient between the values of RIA and bioassay was 0.69 (p<0.01) (Fig. 10).
The urinary FSH levels were determined daily in two normal women throughout the menstrual cycle. Early follicular rise, pre-ovulatory peak and pre-menstrual rise of urinary FSH were observed (Figs. 13 and 14). The randomly voided urines were obtained from 50 pregnant women at random gestational weeks. There was a slight increase in mean FSH concentration between weeks 5-8 and 13-16 of gestation. The mean FSH concentration progressively increased from weeks 25-28 to 33-36 and decreased slightly at weeks 37-40 (Fig. 15). The urinary excretions in a pregnant woman were serially determined throughout pregnancy (Fig. 16). Her periods were almost regular with anovulatory bleeding and she became pregnant by treatments with clomiphene citrate. Urinary FSH excretions exhibited a small peak at weeks 11 to 13 of gestation and increased again during the third trimester. A rapid fall of urinary FSH excretion occurred before delivery.

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In order to study the real ultrastructural changes and the hormonal regulation in the process of the implantation, scanning electron microscopic observations were made on the endometrial surface of delayed implantation and castrated rats.
1) In delayed implantation, many large cellular protrusions, dense arrangement of microvilli and several microapocrine-like structures were observed.
2) Around eight hours after the injection of estradial, which dose was enough to induce the implantation, features such as a lot of macroapocrine-like and microapocrinelike structures, bulging of the cellular surface suggested the increase of secretion.
Subsequently, the decrease of the secretion activity was implied from such features as wrinkling of the cellular protrusions, shortening or bending of the microvilli, and flattening of the cellular surfaces.
3) In the castrated rats, the microvilli were short and sparsely distributed.
Under the administration of progesterone in these rats, microvilli were arranged densely and a large cellular protrusion was distributed on every cell.
In contrast, at twelve hours after the administration of estradiol in these rats, many microapocrine-like structures were observed.
Moreover, under the sequential administration of progesterone and estradiol, these findings well resembled those with estradiol injection in delayed implantation.
4) The significance and relation of those ultrastructural changes with the ovum-implantation were dicussed.

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