We have reported that goat semen diluted with egg yolk citrate buffer solution coagulates in several days of preservation at 4°C, and a sudden drop of pH accompanied the coagulation regardless with lactic acid accumulation. And it is probable that the coagulating factor acts to the egg yolk constituents such as phospholipids, and releases some acids which cause the drop of pH. In this experiment, we attempted to examine the occurrence of phosphorylation and the fatty acids release in the reaction mixture of egg yolk buffer solution and the coagulating factor. Principal results obtained are as follows.
1. It was found that the acid soluble phosphorus was released partially from the phospholipids of the egg yolk only when the seminal plasma was used as a coagulating factor, and not when the Cowper's gland extract was used. Considering these facts, it is likely that phosphorylation is not necessarily accompanied by the coagulation phenomenon, but may be due to other enzymes present in seminal plasma of goat semen.
2. There was a marked increase of fatty acids which may have been released from the egg yolk, when either the Cowper's gland extract or the seminal plasma was used as a coagulating factor, and the former has an about 10 times stronger activity in the releasing action than the latter.
3) There was also an increase of neutral fats in the coagulated egg yolk buffer solution due to the released fatty acids.

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In experiment reported here, we attempted to clarify the position of yolk constituents which would be attacked by the coagulating enzyme and the biochemical changes in the egg yolk constituents when coagulation occurred. Furthermore, it was also one of the aime of this experiment to obtain some knowledges on the nature and the kind of this enzyme. Principal results obtained are as follows.
1. We have examined the fatty acid release in the reaction mixture containing each of the yolk fraction fractionated with organic solvents to confirm the position of the egg yolk constituents from where the fatty acids were released. As the results, fatty acid production was observed only in the reaction mixture containing whole yolk, yolk powder or crude phospholipids, and not in the mixture containing neutral fats or yolkprotein. From these results it was suggested that the coagulating enzyme acts to the ester linkage with acyl group of the egg yolk phospholipids.
2. Paperchromatographic studies were performed on the released fattyacids and the choline-lipids in the coagulated yolk buffer solution. As the results, it was found that the fatty acids released were mainly palmitic acids, stearic acids, and unsaturated acids (oleic acid, and linoleic acid). It was also clarified that the spotting of the lysolecithin was clearly detected on the chromatogram of the cholinelipis. The production of the lysolecithin may be due to the release of the fatty acids from the egg yolk phospholipids, lecithin.
From these results obtained above, it was presumed that the coagulating enzyme was a kind of the 'Phospholipase-A' similar to those of the snake venom active to the ester linkage with acyl group of the egg yolk phospholipids. Now it should be a notable fact that the snake venom acts to the phospholipid and releases only the unsaturated fatty acids and produces lysolecithin in contrast to that the egg yolk-coagulating enzyme releases both of the saturated and unsaturated fatty acids and produces lysolecithin.

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Existence of the egg yolk-coagulating enzyme in goat semen was found recently, yet its purificationhas not been attempted. While it is generally very important and significant to extract an enzyme inthe purified state in order to clarify the characteristics especially for those discovered such as the onediscussed here.
In this experiment, the authors tried to purify the enzyme by the following methods; salting out byammonium sulfate, selecting adsorbtion with tricalcium phosphate-gel, andthe acetone fractionation.
Principal results obtained were as follows.
1. Selecting precipitation method with ammonium sulfate was tried on the goat seminal plasma toobtain the enzyme rich fraction, but most ofthe seminal plasma protein was abruptly salted out ataround 30%ammonium sulfate. And it was difficult to extract the enzyme rich fraction by this method.
2. Then the selecting adsorbtion method was performed. Seminal plasma protein was salted out adding equal volume of saturated ammonium sulfate, and dialized the precipitated protein for 12 hours at 4°C, then it was adsorbed to the tricalcium phosphate-gel, and the selecting elution was made addingvarious concentrations of the phosphate buffer to the protein adsorbed calcium-gel. As the results, considerably purified enzyme was obtained from the fraction eluted into 0.25 M phoshate buffer. Besides, the purity of the enzyme in this fraction was about 10 times higher than that of the starting material, seminal plasma.
3. Purification of the coagulating enzyme was performed by the acetone fractionation starting from the goat seminal plasma. Acetone was added to the goat seminal plasma to be 33%, 45%, 51%, 62.%, 67%, and 77% of the concentration in the mixture at 0°C, then each of the precipitated fraction wasobtained after ultracentrifugation. Thus, it was confirmed that the fraction precipitated at the concentration of 33%acetone shows the highest apecific scivity, and the purity of the enzyme was about 4 times higher than that of the starting materia1, goat seminal plasma.
Furthermore, paperelectrophoretic studies were performed on each of the fraction obtained above. Andit was clarified that the enzyme-rich fraction shows low mobility, and the remaining 4 fractions free from most of the enzyme have high mobility.

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The biological properties of PMSG are thought to be similar to those of the unfractionated pituitary compound of FSH and ICSH. However, it was proved in the present study that effects of PMSG on immature male reproductive organs is not identical with the pituitary GTH.
The Wistar-Imamichi rats8) weighing 28-40 g at the age of 21-23 days were hypophysectomized through parapharyngeal route. The same assay method reported before6) was used. The results was compared with those obtained by ICSH6) and HCG7) by means of the prostate units (the universal biological unit for comparing the activity of gonadotrophins derived from any source).
Testicular weight increased at the small dose (1.43Pr. U.) of PMSG and plateaued at total dose of about 300 Pr. U. ICSH was slightly effective than PMSG on testicular weight, although ICSH did not give to attain the plateaued weight. Tested by unfractionated sheep pituitary GTH (Vetrophin) however, testicular weight initiated to increase at small dose than ICSH and plateaued on the same level as PMSG at smaller dose (32-64 Pr. U.).
Accordingly, the effect of PMSG to increase the testicular weight of 21 day old rats is weaker than that of the pituitary GTH. In all of these cases, the interstitial cells were stimulated, but the seminiferous tubles were differentiated at only the stage of spermatocyte formation. Accessory organs (epididymides, seminal vesicles and ventral prostate) were extremely stimulated by PMSG as well as HCG. Their plateaued weights were obtained at relatively small doses (about 3 Pr. U. in epididymides and about 20 Pr. U. in seminal vesicles and ventral prostate). By ICSH treatment, the seminal vesicles did not stimulated at higher dose (50 Pr. U.), however, at the more dose of the unfractionated sheep pituitary GTH (about 250 Pr. U.) the weight of seminal vesicles increased to the highest levels in the cases of PMSG and HCG treatment. Similarly, the ventral prostate did not responded conspicuously to ICSH. The weight of ventral prostate of animals received more dose of ICSH (50 Pr. U.) was almost twice of hypophysectomized controls, i. e., the same as animals treated with 1 Pr. U. In this case, higher doses of the unfractionated sheep GTH (about 125-250 Pr. U.) increased the weight of ventral prostate up to the same levels as PMSG and HCG (4-5 times of controls).
It may be said that the distinct difference of PMSG to pituitary GTH is the effect of androgen secretion. The stimulative effect of pituitary GTH to secret the testicular androgen is not conspicuous in immature animals. On the contrary, PMSG having the specific properties of the placental GTH like HCG acceralates powerfully the androgen secretion not only in adult but in immature animals. These differences would be accounted for the intrinsical difference, but not the varied rate of content of FSH and ICSH.

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Ovulation inducing activity of unfractionated sheep pituitary gonadotrophin (Vetrophin) was compared with that of HCG.
One rat ovulating unit3) of Vetrophin was 1/26.7 R. U. by subcutaneous administration and 1/256 R. U. by intravenous injection. The ovulation inducing potency of intravenous injection was about 9.5 times active than that of subcutaneous injection. The ratio of effectiveness of subcutaneous and intravenous injection is almost the same as the results obtained by HCG6).
The potency of Vetrophin is shown by Rat Unit which is determined by the ovarian weight stimulating effects. However, based on the rat ovulating unit, 1 R. U. of Vetrophin coresponds to about 180 to 280 I. U. or 60 to 70 M. U. of HCG by subcutaneous injection and to about 370 I. U. of a preparation of HCG by intravenous injection.
It is said that in terms of HCG tested on the increment of ovarian weight in immature rats, Vetrophin provides the equivalent of 2, 000 I. U. in the 1 R. U. dose8, 11). However, compared by the ovulation inducing activity, 1 R. U. of Vetrophin was equivalent to only about 200 or 400 I. U. of HCG by subcu-taneous or intravenous administration respectively. Vetrophin is used mainly by the intravenous injection for the treatment of the reproductive disorders of domestic animals. On the contrary, HCG is usually not administered intravenously. Considering our results, it should be said that the effect of 1 R. U. of Vetrophin (intravenous injection) to treat the cystic ovaries may be as same as the effect of about 2, 000 I. U. or about 1, 000 M. U. of HCG (subcutaneous injection). The results presented here will furnish some information to clinical use of Vetrophin. Finally, we suggest that in the treatment of cystic ovaries, Vetrophin may be used effectively to treat the cases that antihormone against HCG is produced by previous injections of HCG.

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