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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凍結-融解の過程における精子生存の機構を吟味する一環として,グリセリン無添加の卵ク液および脱脂乳中の牛精子の耐凍性を検索し次の知見を得た。
1) 現行の冷却速度(凍結まで1°C/min.以後5°C/min.)では,凍結温度の低下につれて,精子の生存率は急激な低下を示し,卵ク液では-25°Cで死滅する。牛乳の場合,卵ク液に比し生存率が良好で-30°Cでも死滅しない。
2) 上記の速度で凍結した精液を,液体窒素に浸漬する方法で急速な冷却を行なつた場合, -196°Cの超低温下でも生存精子が認められる。
3) この方法による液体窒素中の精子の生存曲線は,いわゆるプラトーの終末点からの急冷で,急に上昇し始め, -15°C(卵ク液) -15~25°C(脱脂乳)でピークに達し,以後急激に低下し,卵ク液では-25°Cからの急冷で死滅する。脱脂乳の場合, -30°Cからの急冷でも低率乍ら生存する。
4) 上記液体窒素中の精子の生存能力は,予備凍結の過程で起こる細胞の脱水の結果として,始めて獲得するものと解釈される。
5) この実験で凍結下における精子の死滅は,既に言われているように,細胞外凍結の過程で起きる塩の濃縮脱水および細胞内凍結の結果によるものと想像され,また2次凍結のさいの死滅は,その冷却速度から見て,細胞内凍結に原因するものと推察される。
本研究は農林水産技術会議による国内留学の規程により,北海道大学低温科学研究所で実施した。研究の企画,指導ならびに原稿作成に御高示を頂いた北大,低温研,根井外喜男教授,留学の機会を与えられた農技研丹羽太左衛門博士,留学中格別の御配慮を頂いた技術会議,道家調査課長,大貫技官,精液の分譲その他積極的な御協力を頂いた北海道道立家畜人工授精所亀松二前所長始め所員の方々の御厚意ならびに低温研第4部門の室員の方々の御協力に深謝する。

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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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以上種々の体液細菌等について観察した牛精子の凝集現象を要約すると次の様である。
1. 野外雌牛血清59例中48例に精子凝集が起り,うち36例は20倍稀釈でも凝集し, 100倍を越えるものは22例,200倍を越えるものは15例認めた。
性周期による雌牛血清の精子凝集性に及ぼす差は明らかでないが,精液別にみると若干の差が認められた。これらの血清は56°C 30分加熱しても凝集を発現し,かなり耐熱性である。
2. 子宮洗滌液,頸管粘液,では少数例に極めて軽度の凝集がみられた程度であるが,頸管部を強く刺戟して出血を起させた場合には粘液中に侵入した精子は凝集し精子運動性を著しく阻害した。
3. 精子を凝集させる大腸菌はKAUFFMAN標準大腸菌20株中9株にみられ,精子運動性を著しく減退させた。頸管粘液に凝集性大腸菌を添加したものについての精子受容性は不良で,侵入性~運動性共に著しく妨げられた。大腸菌による凝集は個体別精液では,差が認められないが,死精子は凝集性が不定となり判定困難である。加熱菌による精子凝集性は100°C 30分では減弱した。菌量と精子凝集性の関係は感作15分で菌量0.0313mg/ccまで24時間で0.0039mg/ccまで凝集する。精子濃度は1~2億/cc位が判定しやすい。
4. 尿には精子凝集を発現させる作用はない。
5. 卵胞嚢腫内容液は血清と同様明瞭な凝集が認められた。
6. 去勢牛の副性器分泌液は精子を凝集させるが,該牛にTestosterone投与すると分泌物の性状が改善され,凝集陰性となるが, Estrogenにはその作用は認められない。
7. 牛精子は塩類溶液で凝集しない。
8. 牛精子の凝集は3型に分けられ, head-head agglutination及びtail-tail agglutination, head-tailの混合凝集であってthigmotropismやchemotropismと区別され特異的なものである。

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