The effect of clomiphene on the ovulation induced with PMS treatment was investigated in immature rats.
PMS (3 IU) was injected subcutaneously to rats of 25 days of age at 9 : 300: 00 A. M.. The animals were killed at 74 hrs. after PMS injection and the tubal ova were checked under the dissecting microscope. Clomiphene (clomiphene citrate) was injecuted subcutaneously as a 30% ethanol-saline solution in the dose per 100 g of body weight. Vaginal smear and weight of organs (ovaries, uterus, pituitary) were also examined at autopsy.
Results obtained wereas follows
: 1) When the clomiphene was injected at 12 hrs. after PMS injection, 5 mg and 1.25 mg of clomiphene inhibited the ovulation completely, while 500 μginhibited in the rate of 60 % and 250 μg had no effect on the ovulation (Table 1).
2) Two doses of clomiphene (5 mg, 500 pg) were injected at various times after PMS injection. In the case of 5 mg treatment (Table 2), the ovulation was inhibited in the rate of 6080% at 0 and 2736 hrs. after PMS injection.
The complete inhibition of ovulation with clomiphene treatment was observed during 324hrs. after PMS injection. Following clomiphene injection at 39 hrs. after PMS injection had no inhi-bitory effect on the ovulation. In the case of 500 μg treatment (Table 3), the inhibition of ovulation was observed in the rate of 60% at 3to 24 hrs. after PMS injection. The inhibitory effect of clomiphene treatment was not shown at 27 hrs. after PMS injection.
In many cases that the ovulation occured by the treatment of two doses of clomiphene, the weight of uterus was smaller than that of control (PMS only). On the otherhand, the ovarian and pituitary weights and vaginal smear were not clearly influenced by clomiphene (Table 2, 3).
3) In the case of HCG (10 IU) injection of 54 hrs. after PMS injection to rats which were given clomiphene (5 mg) at 12 hrs. after PMS injection, ovulation was induced in all animals. In the rats of PMS and clomiphene treatment, tubal ovawere found in all animals at 98 hrs. after PMS injection, so that the delay of ovulation for 24 hrs. is recognized (Table 4).
4) When 0.5 μg of estradiol-17 βwas administered at 3 to 6 hrs. after PMS injection to rats in which the ovulation might be inhibited with clomiphene treatment (clomiphene 1 mg: 6 hrs. after PMS injection), the inhibitory effect of clomiphene was completely overcome. However, in these cases the depletion of uterine weight was not restored.
Since it is known that clomiphene competes with estrogen concerning to estrogen receptor in hypothalamus-pituitary system, it is suggested that endogenous estrogen following to PMS treatment plays an important role on the induced ovulation with PMS treatment.

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Fructose, glucose, sorbitol and inositol concentrations were determined on the luminal fluid and tissue in bovine genitalia at various stages of estrous cycle.
The genitalia was obtained from 23 healthy dairy cows and heifers, and 10 beef heifers at slaughter.
Samples collected for analysis were uterine fluid, cervicalmucus, washings of oviduct, and tissues of isthmus of uterine tube, carunclar area, intercarunclar endometrium and cervix.
They were deproteinized by Somogyi's method, lyophilized, trimethylsilylated and subjected to gas-liquid chromatography for carbohydrate assay. The chromatographic separation was carried out on a 5% SE-52 on Shimalite W column (3 m x 3 mm i. d.) by linear temperature-programmed analysis. Column oven was conditioned for a temperature increase of 1°C per minute from 165°C. Detector oven was maintained at 330°C and injection port at 300°C. A nitrogen carrier gas was used at flow rate 60ml/min. The hydrogen flow to the flame jet was 45 ml/min.
The concentration of the materials was estimated using adonitol as an internal standard.
The results obtained were asfollows.
1. The levels of fructose, glucose, sorbitol and inositol in uterine fluid showed a definite variation with the stage of estrous cycle.
The each concentration of four constituents was low at estrus and after ovulatoan, and increased at early luteal stage. The high levels were maintained until onset of the nextestrus. The ratios of the constituent levels at mid-luteal stage to those at estrus were 15 in fructose, 6 in glucose, 346 in sorbitol, 18 in inositol.
The main carbohydrate was glucose at estrus and sorbitol during luteal phase.
2. Fructose, glucose, sorbitol and inositol were also found in the cervical mucus. Their levels were lower than those in uterine fluid at every stage in estrous cycle.
In the tissue of genitalia, sorbitol occurred only in endometrium during luteal stage. Therefore, sorbitol in the cervical mucus might be due to uterine fluid discharged from the uterine cavity.
3. In the oviduct, active secretion of inositol was found out, but secretion of sugars was uncertain.

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The estrogen levels in the blood, uterine and placental tissue of rats were determined during late pregnancy and at parturition.
The results obtained were as follows:
1. Estrogen levels in the blood during late pregnancy were increased rapidly from 15 to 17 days of pregnancy and the variation of values until 22 days of pregnancy was not significant. The remarkable increase occured at parturition.
2. Estrogen contents in the uterine tissue during late pregnancy and at parturition were lower than those of the placental tissue, the variation during experimental period was also not significant. The contents were decreased at parturition.
3. Estrogen contents in the pleacental tissue during late pregnancy were increased as pregnancy progsesses and a dramatic increase was observed at parturition.

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Histochemical demonstrations of phosphorylase and UDPG-glycogen transferase were carried out using hamster eggs at the stage of blastomere-differentiation.
The results obtained were as follows.
1) Each blastomere of the 8-cell eggs 6570 hours after ovulation showed a strong phosphorylase reaction. Just before differentiation, four of the eight blastomeres of each egg exhibited a strong reaction, while the others showed none. In the differentiated 8-16 cell eggs 7175 hours after ovulation, only trophoblasts showed a weak reaction.
2) In most of the 8-cell eggs, each blastomere showed a moderate reaction of UDPG-glycogen transferase, whereas differentiated 8-16 cell eggs and blastocysts did not.

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The effect of male proximity on induced ovulation by progesterone in the 4-day cyclic female mice was studied.
The animals were housed in artificially illuminated animal rooms (light: 5 a.m. to 7 p.m.).
In this experiment, the 4-day cyclic mice which has not been exchanged at a 3-day oestrous cycle by pre-test of male proximity were used.
The female mice were divided into 2 groups. One of them were placed adjacent to male mice with partition of the wire net in a cage. The others were served as a control and male mice were not employed for this group.
Progesterone was once injected subcuteneously into the female mice of the both groups at 4 p.m. or 7 p.m. on the day of diestrous stage and induced ovulation was examined in the following day (the day of expected proestrous stage). The doses of progesterone were 0.130.25 mg or 0.5 mg per mouse at 4 p.m. or 7 p.m., respectively.
Induction of ovulation due to progesterone was more pronounced in the mice approached by the male mice as compared with the control mice (Fig. 1).
The results obtained were comparable to those of the preceding experiment in which ovulation was enhanced by pretreatment with estradiol prior to progesterone administration.
Thus, it is suggested the male proximity might induced the secretion of estrogen in the female mice.

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There is no information on the qualitative and quantitative alteration of uterine fluid in the rat. This experiment was designed to investigate the biochemical alteration of protein in uterine fluid during estrous cycles in the rat.
Adult virgin Wistar rats, weighing from 180 g to 280 g, was used. Uterine fluid (UF) was obtained from the uterus by flushing with 0.8 ml of saline solution. When the uterus was clearly distended with fluid, the accumulated uterine fluid (AUF) was aspirated with tuberculin syringe. Concentration of protein in uterine fluid was determined by the method of DAUGHADAY, using bovine serum albumin as standard protein. Patterns of protein in uterine fluid were analyzed by polyacrylamide gel disc electrophoresis with a 7.5% running gel. The gels were stained with amido black for protein detec-tion and with periodic acid-Schiff reagent (PAS) for carbohydrate detection after the electrophoresis. The protein fractions were measured as areas under absorption peaks from densitometric tracings.
The results obtained were as follows:
1. AUF appeared between 6:00 hr of proestrus and 6:00 hr of estrus. The fluid increased pro-gressively and reached maximum at 22:00 hr of proestrus (see Table 1).
2. The total content of protein in UF was significantly higher in proestrus than that in other stages of estrous cycle (see Fig. 1), and increased to maximum at 22:00 hr of proestrus. Then they diminished at 6:00 hr of estrus.
3. Protein concentration of AUF collected from 6:00 hr of proestrus to 6:00 hr of estrus was lower in this experiment than the results of others (Table 1).
4. Protein bands in densitometric tracing of the serum and UF were detected 15 and 12, respec-tively. Those protein bands were divided to 8 fractions (A to H) according to the Rf values. The pattern of protein in proestrous UF was different from those of other stages resembling to the pattern of serum protein (Table 2). The alteration of their fractions appeared from 6:00 hr of proestrus to 6:00 hr of estrus. It has been obtained that B fraction (albumin) was one quarter and H fraction (γ-glo-bulin) was at least twofold in comparison with those of serum (Table 4).
5. Glycoprotein fractions of serum and UF were shown in Table 3 and 5. Since patterns of glycoprotein were similar to patterns of protein in UF obtained from 10:00 hr of proestrus to 6:00 hr of estrus, it was suggested that much glycoprotein have been contained in these UF than in UF of other stages.

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[in Japanese]

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