[in Japanese]

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An enzymohistochemical demonstration was carried out using rabbit ovaries obtained 10 days after mating from normally pregnant, and pregnant or non-pregnant rabbits after superovulation treatment. The results are as follows.
Corpora lutea from normally pregnant rabbits showed week or very slight hydroxysteroid dehydrogenase (OH-SDH) activities: activities of 3α-OH-SDH (NAD dependent), Δ⁵-3β-OH-SDH (DNA as a subtrate, NAD, NADP ; pregnenolone, NAD), 11β-OH-SDH (NAD), 16β-OH-SDH (NADP) and 17β-OH-SDH (NAD) were weak, while those of 3α-OH-SDH (NADP), Δ⁵-3β-OH-SDH (pregnenolone, NADP), 11β-OH-SDH (NADP), 16β-OH-SDH (NADP) and 17β-OH-SDH (NADP) were weaker, or slight. As for other dehydrogenases (DH), activities of G-6-PDH and MDH (NAD) were intense, those of SuDH and LDH were moderate, of MDH (NADP) and α-GDH were weak, and of β-GDH and GDH were slight. Activity of acid phosphatase was moderate, while that of non-specific esterase (Etase) was weak.
Corpora lutea from superovulated pregnant rabbits exhibited almost the same enzyme activities as those from normally pregnant rabbits, except for the decrease shown in LDH and Etase activities. On the other hand, corpora lutea from superovulated but non-pregnant rabbits showed weaker activities, in the OH-SDH that showed weak but plus activities concerning pregnant rabbits. They showed weaker activities in SuDH, MDH (NAD) and α-GDH also. Blood follicles that appeared in treated rabbits showed similar enzyme activities to corpora lutea.
Interstitial tissue, too, were observed concerning about the same enzymes as above.

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The distribution and quantitative fluctuation of nucleic acids in hamster eggs in early development were investigated through the use of fluoresccence microsopy, using acridine orange as the vital fluorochrome. The results obtained were as follows.
The metaphase chromosomes of unfertilized and penetrated eggs were located in their cytoplasm near the extruded first polar bodies, in which also were found chromosome granules. These chromosomes showed strong greenish yellow fluorescence, indicating the presence of DNA. Penetrated sperm head exhibited a moderate strength of fluorescence. In pronuclear and 2-celled eggs, the nuclear fluorescence decreased. With further development of eggs, from 4-celled eggs to early blastocysts, gradual increase was observed in the strength of fluorescence in parallel with the diminution of their nuclei.
At every stage of development, the cytoplasm of eggs showed red fluoresence, indicating the presence of RNA and mononucleotides: the eggs before pronuclear stage showed strong red fluorescence throughout the cytoplasm, while in 2-celled eggs, the strength of fluorescence somewhat decreased, and showed a tendency to be localized near the nuclei. In 4 to 8-celled eggs, it further decreased, and was localized around the nuclei. In the differentiated 8 16 celled eggs and early blastocysts, trophoblasts and inner cells showed strong red fluorescence throughout the cytoplasm. No fluorescence was observed in the nucleoli of the eggs at any stage.

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[in Japanese]

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The proportion of eggs undergoing fertilization in vitro was compared between intact (Fig. 1, A) and denuded (Fig. 1, B) eggs using capacitated (pre-incubated) spermatozoa.
Superovulated eggs were obtained 1617 hr after injection of HCG from adult female mice of ICR-JCL strain. They were liberated from the oviduct under warm mineral oil in a plastic petri bish containing 0.4 ml of incubation medium (modified Krebs-Ringer-bicarbonate solution). Immediately, one group of eggs with cumulus cells was treated with hyaluronidase by adding 0.08 ml of a hyaluronidase solution (900 unit/ml) to the incubation medium containing egg clots. After incubation for about 7 min, the denuded eggs were transfered through three changes of the medium to remove the cumulus cells. Another group of eggs was not treated with hyaluronidase and used for fertilization immediately after recovery.
The sperms used were recovered from cauda epididymis of adult males and suspended in the incu-bation medium in a concentration of 3, 0006, 000/mm³. The sperms were then incubated at 37°C under 5% CO₂ in air for 1 hr before they were used for insemination.
At the time of insemination, a drop of pre-incubated sperm suspension (ca. 6.310.3 mm³) was added to the medium containing intact or denuded eggs. Final concentration of sperm was estimated at 50400/mm³.
After insemination, the eggs and sperms were incubated at 37°C in a humid atmospher of 5% CO₂ in air for 4 to 5 hr. At the end of incubation, the eggs were placed on a glass slide, compressed gently under a cover slip and examined for sperm penetration through the zona pellucida, sperm entry into the vitellus and the formation of male and female pronuclei.
The results indicate that there is no significant difference in the proportion of penetrated eggs (Table 3), the proportion of eggs undergoing fertilization and the rate of polyspermy (Table 2), whether the eggs are denuded or not. The proportion of eggs undergoing fertilization was 71.3% in denuded eggs as compared with 84.7% in intact ones. The numbers of spermatozoa found in the penetrated eggs also showed similar distribution between two groups.
It is considered from these results that the presence of cumulus cells does not play a role for the sperm penetration of mouse eggs in vitro.

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