In earlier reports of our series of studies, it was demonstrated that the antihormone against HCG (Anti-HCG) might be produced easily in cattle. In this paper we undertook investigations on the reformation of Anti-HCG in cattle by injecting smaller doses of HCG than those used in the previous experiments.
A female calf and a cow with normal estrous cycle and 3 cows with cystic ovaries were used in the experiments. These animals had been received one or more injections of various doses of HCG and their serum Anti-HCG had been determined to be positive approximately 150-200 days prior to the present experiments. The methods to determine the serum Anti-HCG were same as described in previous reports.
The results obtained were as follows.
1. Reformation of Anti-HCG in experimental animals.
A female calf and a cow employed in the present experiments were same as described in report I. After the determinations of serum Anti-HCG produced formerly became negative, the 2 animals were reinjected intravenously with daily dose of 2, 500 MU of HCG for consecutive 4 days (10, 000 MU in total). This mode of experiment was repeated 3 times at intervals of 100 days and 105 days respectively. The results of the determinations of serum Anti-HCG were shown in Table 1 (Cattle No. 1) and Table 2 (Cattle No. 2). The results obtained from the experiments indicated that the Anti-HCG was reformed by reinjecting HCG more readily than that was produced by injecting HCG for the first time. The potencies of the reformed Anti-HCG (HCG inhibiting ability per 1 cc. of serum) about 10 days after the reinjections in these 3 trials were 3.0 MU, 6.0 MU and 12.0 MU in cattle No. 1 and 1.5-3.0 MU, 3.0 MU and 6.0 MU in cattle No.2 respectively. After the 3rd reinjection, the reformed Anti-HCG remained until 100 days in cattle No. 1 and 75 days in cattle No.2, and then disappeared on the 125th day and 100th day respectively. It was observed from these data that the potency of the reformed Anti-HCG increased gradually through the repeated experiments, and the duration of existence of Anti-HCG was longer in the animal having higher Anti-HCG potency than that having lower potency.
In the next experiment, 105 days after the 3rd reinjection, cattle No. 2 was received single injection of 2, 500 MU of HCG in order to examine whether such smaller dose of HCG might induce the reformation of Anti-HCG. Detectable amounts of Anti-HCG appeared on the 3rd day (HCG inhibiting titer per cc. of serum : 1.5 MU) and the reformed Anti-HCG was observed to increase rapidly from the 5th to the 10th day (inhibiting titer: 6.0-12.0 MU/cc.) as shown in Table 3. From these results, it appeared that the reformation of Anti-HCG could be induced rapidly, reached to the maximum level about 10 days after the reinjection, and thereafter fell gradually.
2. Reformation of Anti-HCG in cows with cystic ovaries.
Three cows with cystic ovaries were also employed in the experiments of the reformation of Anti-HCG. They had been received 2-3 intramuscular injections of HCG for treating ovarian follicle cysts. The histories of the HCG treatments and the results of the Anti-HCG determinations in these animals were as shown in Table 4. The initial potency of the serum Anti-HCG (serum No. I) was 24.0 MU or more, 3.0 MU and 3.0 MU respectively in these 3 animals. All of them had recovered from ovarian follicle cysts by intramuscular (Cattle No. 2) or intrafolliclar (Cattle No. 1 and No. 3) injections of HCG, and become pregnant. The reinjections of HCG were held at the time when 2 (Cattle No. 2 and No. 3) of the 3 animals were in about 170 days of pregnancy and the determinations of serum Anti-HCG (serum No. II) being negative, while remaining 1 animal (Cattle No. 1) suffered again from ovarian follicle cyst about 60 days abortion happend on the 89th day of pregnancy and the determination of serum Anti-HCG (serum No. II) remaining positive (inhibiting titer: 1.5 MU/cc.).

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It has been believed that FSH stimulates the follicular growth and does not induce ovulation. However, there were some reports suggesting the posibility that FSH might have the luteinizing and ovulation promoting properties13, 17, 18, 19, 24).
In the present study, the ovulation inducing activity of swine FSH was investigated. Regular 4 day cyclic rats were used. Purified swine FSH was injected subcutaneously at the afternoon on the day of vaginal diestrus. Animals autopsied 20 hours after the injection and the tubal ova were inspected.
At the dose of 50 γ and 100 γ of FSH, 4 out of 10 and 8 out of 10 animals ovulated. From these results, 1 rat ovulating unit16) of this preparation of FSH was calculated as 60 γ.
Generally, the amount of contaminating ICSH in the preparation of FSH is estimated by the ventral prostate assay of hypophysectomized immature male rats. In this preparation of FSH, the amount of contaminating ICSH is estimated as 2-3% by the prostate assay22). As the amount of 1 Pr. U. of sheep ICSH was 40 γ15), 1, 300 to 2, 000 γ of the FSH preparation is equivalent to 1 Pr. U. The amount of 1 R. Ov. U. of the ICSH preparation was 7.5 γ which was equivalent to 0.19 Pr. U. If the ovulation was induced by the contaminating ICSH in the FSH preparation, the necessary amount of this preparation to induce ovulation in 50% of animals may be calculated as 250 to 375 γ. However, the practical amount of 1 R. Ov. U. of the FSH preparation was 60 γ which was equivalent to 0.03 to 0.0045 Pr. U. and its ICSH contamination is calculated as 1. 2 to 1. 8 γ by the ventral prostate stimulation.
By this result, ovulation inducing activity of the FSH preparation is 4 to 6 times potent that of the ICSH preparation. The ovarian interstitial cell stimulating effect of 400 γ of the FSH preparation was weaker than that of 5 to 10 γ of ICSH. Accordingly, ICSH contamination in 60 r of the FSH preparation may not exceed 2 γ. Although 200 γ of the FSH preparation stimulated effectively the follicular growth in hypophysectomized immature female rats18), 21), no follicle was luteinized by combined treatment of 200 γ of FSH and 5 to 1, 000 γ of ICSH17). On the other hand, partial luteinization was observed clearly in hypophysectomized immature animals received 500 γ of FSH alone. From these results, it may be reasonable to assume that the luteinizing and ovulation inducing activities may be intrinsic as biological properties of pituitary FSH.

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The effects of gonadotrophin (HCG) and hypophysectomy on androgen-genesis of rat testis, in vivo were studied. As a precursor, progesterone-4-C¹⁴ was injected intravenously to eviscerated rats under various conditions and radioactive steroids were analyzed qualitatively and quantitatively in the testis, blood and other tissues.
Results obtained are summarized as follows:
1. Relative high concentration of total radioactivity is found in the sexual accessory glands, especially in the preputial gland, however, none of them contains testosterone (Table. 1 & 2.).
2. Testosterone is detected in the testis and blood, not onlyin adult rats but also in the HCG treated 30-day old rats, however, the presumedintermediates from progesterone, ie. 17α-hydroxyprogesterone and androstenedione, are not detectable throughout the experiments (Table. 2 & 3).
4. When theanimal possesses normal testosterone-genesis system (abb. TGS), HCG accelerates it promptly and increases either production and secretion of testosterone or utilization of progesterone. Four weeks after hypophysectomy, production of testosterone ceases and progesterone is scarcely util-ized and found in a high concentration in the testis, due to impairment of TGS. The capacity of immediate response to HCG is completely lost by this time and no testosterone is produced even if a large dosage of HCG is given. Impaired TGS, however, can be restored by administrations of HCG for a considerable long period even in 10 weeks after hypophysectomy, and production and secretion of test-osterone are observed being accompanied with utilization of progesterone. (Table 3.).
4. Among unidentifiedsteroids, a compound designated as [Pregnenolone] is formed from progesterone and found in the most of tissues analyzed. This is separated from Peak A (Fig.. 1) by acetylation and moves to a less-polar region than pregn-4-ene-20-α-ol-3-one-acetate. Its mobility is identical to that of authentic pregnenolone-acetate, applied as the internal standard. Heretofore, the reaction between pregnenoloneto progesterone is regarded as a irreversible one. Therefore, further careful characterization is needed for this substance.

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[in Japanese]

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