Effects of sugar, glycine, milk and egg-yolk in the calcium-free Krebs Ringer-phosphate on the liability of dog spermatozoa stored at 4°C were studied.
1. It was indicated that the calcium-free KRP buffer was less available on the storage of dog spermatozoa and the longest survival period of dog spermatozoa were seven days in treatments.
2. The sugers were less effective on the viability of diluted dog spermatozoa in the calcium-free KRP buffer.
3. Egg yolk-calcium-free KPR buffer was superior to the milk-calcium-free KRP buffer improving for the storage of dog spermatozoa.
4. Glycine was seemed to harmful on the survival of dog spermatozoa without egg-yolk extenders.

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The composition of semen extenders was studied for the strage of dog spermatozoa at 4°C.
The results obtained are as follows:
1. The basic buffer containing magnesium chloride, potassium chloride, sodiummonohydrogen phosphate and sodium chloride was used. Extenders were composed by adding 20 percent egg-yolk or 50 percent milk to the basic buffer.
It was found after the systematic studies with dog semen extended 1 : 9 and stored at 4°C that. the best motility index after dilution was 74, and that survival period was three days in Experiment I. Survival of dog spermatozoa in the sodium citrate extender was not so longer, though survival period was prolonged by four days.
2. The basic extender in Experiment II was contained with potassium chloride, sodiummonohydrogen phosphate and sodium citrate. Dog spermatozoa could survive for the longest period in the extender added by 1g of peptone, 0.75g of glycine and 10% of eggyolk to the 100 ml of the basic buffer. The longest survival period was 16 days in Experiment II, but motility was decrined to a half after one day.
3. The effect of addition of chlorpromazine for the storage of dog spermatozoa was examined in Experiment III with the extender of Experiment II. The survival of dog spermatozoa stored in extender containing chlorpromazine in concentration of 5 mg/100 ml was prolonged than in the extender containing no chlorpromazine.

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Morphological observation was made on the kind of blood follicles developed by the superovulation treatment in rabbit ovaries and on the changes of the follicles during the course of atrophy. Following is the results:
1. In the ovaries immediately after the superovulation treatment, a lot of well-developed follicles and blood follicles appeared, the latter showing different degrees of hemorrhage. Well-developed follicles disappeared within 7 days after the treatment, while blood follicles remained as long as 30 days after the treatment.
2. Histologically, the blood follicles were divided into two types: cystic blood follicles and luteinizing blood follicles. Immediately after the treatment, there also appeared blood follicles having intact granulosa cells, which seemed to be transformed into either cystic or luteinizing follicles. Luteinizing blood follicles existed in the ovaries up to 15 days after the treatment, and then were transformed into hemosiderin-deposited interstitial glands. The hemosiderin pigment in these glands disappeared completely within 60 days after the treatment. Only a few interstitial glands existed in the ovaries just after superovulation treatment, through gradually increased thereafter. Follicular development was not observed.
3. Hemorrhagic erythrocytes gradually grew sudanophilic and PAS-reactive. The hemosiderin pigment at first appeared in the antrum near follicular membrane, but then infiltrated into perifollicular tissue. Accompanying the formation of interstitial glands, most of the hemosiderin pigments were deposited near the center of the interstitial gland. Hemosiderin-bearing interstitial glands contained more neutral fats and cholesterols than normal ones.

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Recently, it has been demonstrated by authers that the single subcutaneous injection of progesterone (0.25 mg and 0.5 mg/mouse) at 7 p.m. on the day of stage V induced the ovulation as a high rate on the next day, and the induced ovulation by progesterone (0.5 mg/mouse) was observed 135 hrs. after the injection (at 7 p.m. on the day of stage V) in the 4-day cyclic mouse (IV CS strain).
In this study, induction of the ovulation is tried by injection of estradiol on the some times during days of stage IV and V in the 4-day cyclic mice (IV CS strain) under the constant lighting from 5 a.m. to 7 p.m.
The results obtained are as follows:
1) In the 4-day cyclic mice, the single subcutaneous injection of estradiol (0.1 μg and 1 μg/mouse) at 10 p.m. on the day of stage IV (metestrus) induced the ovulation on the day expected stage I (proestrus) as a high rate (Fig. 1, 2, 4 & 5).
2) Induced ovulation by estradiol (1 μg/mouse) was observed 46 a.m. on the expected stage I (27-29 hrs. after the injection), it was injected singly and subcutaneously at 1 a.m. on the day of stage V (diestrus) in the 4-day cyclic mice (Fig. 3).

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Studies on metabolism of amino acid and synthesis of protein in rat embryo have done in our laboratory ¹³⁾. Thus, it is necessary to investigate the relationship between incorporation rate in embryo and content of free amino acids in the uterine fluid under various conditions.
In this paper, free amino acids in the rat uterine fluid in sexual cycle and under various hormonal. conditions are reported.
Adult female rats of Wistar Strain weighing from 180 to 250 g were used. Uterine fluids were collected from seven groups as follows: Group I-Natural proestrus, killed on 2 P.M., Group II-Natural estrus, killed on 9 P.m., Group III-The cervix was ligated through 2-cycle, Group IV-Pseudopregnancy, Group V-Castration and estrone injection (4 μg/day), Group VI-Castration and pro-gesterone injection (4 mg/day) and Group VII-Castration, estrone (1 μg/day) and progesterone (4 mg/day) injection. Each group consists of 24 rats. The schedules for the experiment are shown in Fig. 1. The accumulated uterine fluids were aspirated with a sterilized syringe. When the uterine fluids were-so little, the uterus were washed repeatedly with 0.2 ml of sterilized physiological saline and then. the washed fluids were aspirated. The aspirated fluid was recorded the volume immediately (Table 1). The fluids collected were centrifuged and stored at -20°C until the measurement. These fluids.. were deproteinized with ethanol (final conc. 75%) and then were centrifuged and the supernatant were distillated under diminished pressure. The amino acid was determined with amino acid auto-analyzer. In Group IV, the measurement was divided to two subgroups : the accumlated fluid and washed fluid.
The amount of each uterine fluid is in agreement with the results^{13, 14)} reported previously. The sixteen amino acids of uterine fluid were identified in all groups (Table 2). The content of glutamic acid was the highest value in all group. On the other hand, it has reported that glycine was the highest amino acid in rabbit. Contents of glutamic acid, alanine, threonine and glycine was similar in group IIII. The increase in the contents of methionine and threonine was observed in pseudopregnancy (group IV). These amino acids seemed to be important for blastocyst development²⁰²²⁾. The results of these experiments demonstrate the effect of hormonal status of the animal on theuterine secretions. Threonine, alanine, argine, glutamic acid, leucine, serine and other amino acids have increased in progestational stage as compared with proestrous and estrous stage.
Free amino acids in intact animal's serum were measured in proestrus, 5th day and 14th day of pregnancy (Fig. 2).
Argine, threonine and glycine in serum decreased markedly according to stages of pregnancy. It seemed that there was no relation between the increase of free amino acid of uterine fluid and the decrease of free animo acid in serum during pregnancy.

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Adult female mice of ICR-JCL strain were superovulated by intraperitoneal injections of PMS and HCG 48 hr apart. They were killed 1617 hr after injection of HCG and their eggs were recovered under warm mineral oil in a plastic petri dish containing 0.4 ml of incubation medium. Sperm suspension was prepared by suspending a dense mass of sperms recovered from cauda epididymis of adult male into 0.4 ml of incubation medium under mineral oil. The incubation medium was a modified Krebs-Ringer Bicarbonate solution containing glucose, sodium pyruvate, bovine albumin and antibiotics. (Table 1)
At the time of insemination, a drop of sperm suspension (ca. 510μl) was added to the medium containing egg clot. Final concentration of sperm was 100500/mm³.
The percentages of penetrated eggs were 0, 22.8, 96.4, 93.9, 90.9 or 92.7%, when examined at 1/2, 1, 2, 3, 4 or 5 hr after insemination, respectively (Table 2). Thus, in this experimental condition, sperm penetration of zona pellucida starts arround 1 hr and completes within 2 hr after insemination. At 2hr after insemination all the fertilized eggs showed enlarged sperm head and anaphase or telophase of second meiosis, while at 5 hr after insemination, most of them were found to have male and female pronuclei. Average number of sperm per penetrated egg was 1.6 (Table 3) and the rate of polyspermy was 4.1%.

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Capacitation in vitro of mouse spermatozoa was studied by examining the time of in vitro penetration of eggs by previously incubated sperm.
Sperms recovered from cauda epididymis of adult males of ICR-JCL strain were suspended in a chemically defined medium (Krebs-Ringer-Bicarbonate solution supplemented with glucose, Na-pyruvate, bovine albumin and antibiotics) and incubated at 37°C under 5% CO₂ in air for various period before they were used for insemination.
When sperms incubated for 2 hr were used for insemination, the rate of sperm penetration through the zona pellucida, as examined at 1/2, 1, 2, 3, or 4 hr after insemination, was 57.1, 100.0, 100.0, 98.9 or 98.7% respectively, in comparison with 0, 22.8, 96.4, 93.9 or 90.9% for fresh epididymal sperm (Table 1 and Fig. 1). In the former group, 96% of eggs examined at 1 hr after insemination were at telophase of second maturation division with enlarged sperm head(s) in vitellus, while in the latter group, only 10.9% of eggs were found to be fertilized at this time. It is, thus, clear that in-cubated spermatozoa penetrate the eggs sooner than the fresh ones. The mean number of penetrating sperm per penetrated egg was also higher (3.1) in eggs inseminated with incubated sperm than that (1.6) in eggs with fresh sperm (Fig. 2).
When the eggs were inseminated with spermatozoa which had been incubated for 37, 15, 30, 60 or 120 minutes, 24.1, 70.1, 93.9, 98.7 or 100% of eggs were found to be penetrated, and 5.7, 46.7, . 73.7, 89.5 or 96.0% of them were found to be fertilized at 1hr after insemination (Table 2). This indicates that the change occurred in sperms during incubation is of a progressive nature and is a fairly rapid one, virtually completing within 1 hr after the start of incubation.
These results seem to show that mouse epididymal spermatozoa can be capacitated in vitro in a chemically defined medium without the presence of female reproductive tissue fluid. This will favor the view that capacitation, at least in mouse, is dependent on some appropriate physico-chemical environment rather than on some specific substances from the female reproductive tissues.

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