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[in Japanese]

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ETO and IMAMICHI (1955) reported that ova had been observed in the oviduct inserted betweentwofold glass slides by the dissecting microscope at 20 hours after a single subcutaneous injection of HCG in a diestrous adult female rat. This finding was significant for the bioassay of gonadotrophin.
The authors showed that the sensitivity induced ovulation by HCG was elevated in the case of intravenous injection, as a result of comparison between s.c., i.p., and i.v. injection. Moreover, ovulation was induced 10 hours after injection of HCG (more than 2IU) and ova were observed in the oviduct 12 hours after injection.
This report is concerned with the interval between the induced ovulation by intravenous injectionof HCG (injection being done at 4-5 PM on the day of diestrous stage) and spontaneous ovulation thereafter and with the fate of ova ovulated by HCG injection in adult rats.
The following results were obtained.
1) The interval between the induced ovulation and the first spontaneous ovulation thereafter (72hours) was 3 days, which was shorter than 4 days (96 hours) that is the interval between two spontaneous ovulations in the normal oestous cycle.
2) In the ovulation induced by such an amount of HCG as sufficient to induce ovulation, no tubal ova were observed during 14 and 24 hours before the first spontaneous ovulation after the induced ovulation. In the normal oestrous cycle, no the ova by spontaneous ovulation was observed during 24 and 34 hours before the next ovulation.
Namely, in normal-cyclic rats, ova remained in the oviduct for about 72 hours after spontaneous ovulation, and in the case of induced ovulation ova remained in the oviduct about 58 hours after the induced ovulation by HCG injection.
3) In the first spontaneons ovulation after the induced ovulation by HCG, an ovulation-blockade by pentobarbital (nembutal) anesthesia was seen when the animal was anesthetized during a period from noon to 2 PM on the day before the ovulation, but it was not seen when the animal was anesthetized during a period from 4 to 6 PM on the day before the ovulation.
4) In the first oestrus after the induced ovulation by HCG, the accumulation of uterine fluid was smaller in amount than in the normal proestrous stage.
5) When examined by the vaginal smear method, the animal with the estrous cycle of induced ovulation by HCG showed stages I, IV, II, and III, but the animal in which no ovulation had occurred showed stages I, III, IV, and V in the same manner as the animal with the normal oestrous cycle.

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The method of assy for HCG using ca. 21 days old dd mice to the mouse unit and the interna-tional standard is investigated. The results may be summarized as follows:
1) The difference between body weight at the time of a subcutaneous injection with 2.0 MU HCG using the assay based on hemorrhagic follicles and corpora lutea formation in ovaries of 21-day-old immature female dd mice is significant, the positive response in the range of body weight 8.0-8.5 g and 9.0 g being greater.
2) The difference between right and left ovary in each body weight group is not significant. The result suggests that either ovary should give satisfactory assay results for HCG.
3) Using the weight range of around 8.09.0 g dd mice, the positive response to HCG being greater, the positive reaction rate shows a tendency to increase with increasing dosage of hormone at dose levels 0.5, 1.0, 1.5, 3.0, 4.5, 6.0 and 8.0 IU below 1.0 IU the reduced response is observed. A plateau slope covers the dose above 4.5 III, though the significant difference is obtained except the dose range 0.5:1.0, 4.5:6.0, 4.5: 8.0 IU groups.

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Maturation and time of ovulation of oocytes in adult rats treated with gonadotrophin were studied. A total 239 rats were kept under artificial lighting conditions (night controlled from midnight to 10 a. m. for 10 hours), and killed at various intervals after the beginning of artificial night or HCG injection. At autopsy, ovulated ova were counted, as well as ova recovered from the uterine tubes. For cytological observation of the oocytes, the ovaries of the same rat were fixed in Bouin's solution and stained with Heidenhain's iron haematoxylin. Oocytes of various stages derived from the follicles about 300μ and over in average diameter were examined. The results obtained are as follows.
1. When rats were examined at autopsy 8 to 81/2, 91/2 to 101/2, and 111/2 to 141/2 hours after the beginning of artificial night, it was proved that the average number of ovulated ova was 9. 4, 10.4, and 10.9, respectively.
2. When rats were injected with 25 iu of HCG and examined 10 (6), 12 (8), 14, (10), 16 (12) hours after HCG injection (after the beginning of artificial night in parentheses), the average number of ovulated ova was 2.7, 9.0, 10.6, and 11.0, respectively, and the number of maturing oocytes at various stages, including transitional phase to prometaphase I, was 10.7, 4.4, 3.1, and 2.1, respectively.
3. When rats were injected with 50 iu of HCG and a priming dose of 50 iu of PMS, and killed 10 (6), 12 (8), 14 (10), 16 (12), and 18 (14)hours after HCG injection (after the beginning of artificial night), the average number of ovulated ova was 1.0, 8.5, 19.2, 35.6, and 46.9, respectively, and the average number of maturing oocytes at various stages was 49.5, 41.7, 24.7, 7.1, and 1.6, respectively.
4. Of the 49.5, 41.7, and 24.7 oocytes found 10, 12, and 14 hours, respectively, after the treatment with 50 iu of HCG and a priming dose of 50 iu of PMS, 23.4 (47.3%) of the 49.5 were in telophase I, 34.5 (82.7%) of the 41.7 in metaphase II, and 21.7 (87.9 %) of the 24.7 in metaphase II.
5. When rats were killed at various intervals after HCG injection, the number of degenerating oocytes was 13. 0 to 18.4 in ovulated, and 2.4 to 9.0 in superovulated rats. Total number of ovarian oocytes and ovulated ova was 60.2 to 99.7 per rat in both treated groups.
6. From the results mentioned above, it is suggested that the maturation and ovulation of oocytes may progress in the same pattern in superovulation-treated adult rats as in ovulated rats. Complete ovulation, however, occurred in those rats a few hours later than in the ovulated rats because of development of many oocytes in the former. From cytological observation, it seems that most of the oocytes showing delayed maturation at various times after treatment with HCG alone or with PMS and HCG may degenerate before ovulation takes place.

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Relationships between the antiserum to bovine luteinizing hormone (LH) and the extracts of bovine organs or the hormones of anterior pituitary were studied by the hemagglutination and the hemagglutination-inhibition techniques, in relation to immunoassay of LH.
Antiserum to bovine LH preparation (NIH-LH-B₃) was obtained with rabbit. The antiserum agglutinated the tanned cells sensitized with bovine LH, bovine TSH and bovine GH, but did not agglutinate the cells sensitized with ovine FSH, bovine prolactin and ACTH of unknown species.
The antiserum absorbed with bovine serum and bovine TSH did not agglutinate the tanned cells sensitized with the anterior pituitary hormones except LH. Hemagglutination titer of the antiserum absorbed with bovine serum and bovine TSH against bovine LH was 1: 2560.
The hemagglutination reaction of the tanned cells sensitized with bovine LH and the antiserum to bovine LH absorbed with bovine serum and bovine TSH was not inhibited by the extracts of brain, liver and spleen, but inhibited by the extract of anterior pituitary.
As to anterior pituitary hormones, ovine FSH, bovine GH, bovine prolactin and ACTH of unknown species preparations did not inhibit the hemagglutination reaction, but bovine LH and bovine TSH preparations did. The cause of inhibition of hemagglutination reaction by bovine TSH may be due to LH contaminated in TSH preparation, since the bovine TSH preparation used in this study was not a pure antigen for immunological studies.
The influence of the amount of bovine LH used in the sensitization procedure on the sensitivity of hemagglutination reaction: the tanned cells sensitized with the LH solution more than 0.2 μg/ml were agglutinated by the antiserum, but not agglutinated in the case of less than 0.1μg/m/. In the range of the concentration of LH solution from 0.2 to 50.0 μg/ml, there was no relationship between the sensitivity of the hemagglutination reaction and the concentration of LH solution.
The influence of the amount of LH used in the sensitization procedure on the sensitivity of the hemagglutination inhibition reaction: in the range of the concentration of LH solution from 0.2 to 50.0 μg/ml, the highest sensitivities were obtained at the concentrations of 0.2 and 0.78 μg/ml and thesensitivity of hemagglutination inhibition reaction showed a reverse tendency to the increase of the concentration.Acknowledgement The authors wish to express their gratitude to the Endocrinology Study Section, National Institutes of Health, U.S.A. for the gift of the anterior pituitary hormone preparations.

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